Comparative phenotype analysis of two or more microorganisms using a plurality of substrates within a multiwell testing device

ABSTRACT

The present invention relates to growing and testing microorganisms in a multitest format which utilizes a gel forming matrix for the rapid screening of clinical and environmental cultures. The present invention is suited for the characterization of commonly encountered microorganisms (e.g., E. coli, S. aureus, etc.), as well as commercially and industrially important organisms from various and diverse environments (e.g., the present invention is particularly suited for the growth and characterization of the actinomycetes and fungi). The present invention is also particularly suited for comparative analysis of phenotypic differences between cell types, including strains of microorganisms that have been designated as the same genus and species, as well as other cell types (e.g., mammalian, insect, and plant cells).

The present application is a Continuation-in-Part of U.S. patentapplication Ser. No. 08/762,656, filed Dec. 9, 1996, now U.S. Pat. No.5,882,822, issued Mar. 16, 1999, which is a Continuation-in-Part of U.S.patent application Ser. No. 08/421,377, filed Apr. 12, 1995, now U.S.Pat. No. 5,627,045, issued May 6, 1997.

FIELD OF THE INVENTION

The present invention relates to growing and testing any cell type in amultitest format which utilizes a gel forming matrix for the rapidscreening of clinical and environmental cultures. The present inventionis suited for the characterization of commonly encounteredmicroorganisms (e.g., E. coli, S. aureus, etc.), as well as commerciallyand industrially important organisms from various and diverseenvironments (e.g., the present invention is particularly suited for thegrowth and characterization of the actinomycetes and fungi). The presentinvention is also particularly suited for analysis of phenotypicdifferences between strains of organisms, including cultures that havebeen designated as the same genus and species. In addition, the presentinvention provides methods and compositions for the phenotypic analysisand comparison of eukaryotic (e.g., fungal and mammalian), as well asprokaryotic (e.g., eubacterial and archaebacterial) cells.

BACKGROUND OF THE INVENTION

Ever since the golden age of microbiology in the era of Koch andPasteur, methods for identification of microorganisms have beeninvestigated. Koch's experimental proof that microorganisms causedisease in the early 1800's, provided the impetus to study methods togrow and characterize harmful, as well as beneficial microorganisms.Koch's early experiments to determine the etiology of infectiousdiseases, led to methods for cultivation of microorganisms on thesurface of solid media (e.g., potato slices, see Koch, "Methods for theStudy of Pathogenic Organisms," in T. D. Brock, Milestones inMicrobiology, American Society for Microbiology, 1961, pp. 101-108;originally published as: "Zur Untersuchug von pathogenen Organismen,"Mittheilungen aus dem Kaiserlichen Gesundheitsamte 1: 1-48 [1881]).These studies eventually led to the development of agar as a culturemedium component useful for producing solid media for growing isolatedcolonies of bacteria. To this day, isolated colonies are required (i.e.,"pure cultures") to biochemically identify organisms.

The field of diagnostic and clinical microbiology has continued toevolve, and yet, there remains a general need for systems that providerapid and reliable biochemical identifications of microorganisms. Inparticular, it has been very difficult to develop an identificationsystem which is capable of identifying various diverse types oforganisms, ranging from the common isolate Escherichia coli to the lesscommonly encountered actinomycetes and fungi.

Methods and identification systems to characterize microorganisms widelyused in industry for production of food and drink (e.g., beer, wine,cheese, yogurt, etc.), the production of antibiotics (e.g., penicillin,streptomycin, etc.), bioremediation of oil spills, biological control ofinsect pests (e.g., Bacillus thuringiensis), and the production ofrecombinant proteins, are still needed. In addition, very fewidentification methods and systems have been developed for environmentaluse and there remains a need for simple and generally usefulidentification methods of many organisms. In particular, methods foridentification and growth of the actinomycetes are lacking.

I. The Actinomycetes

The actinomycetes (members of the order Actinomycetales) include a largevariety of organisms that are grouped together on the basis ofsimilarities in cell wall chemistry, microscopic morphology, andstaining characteristics. Nonetheless, this is a very diverse group oforganisms. For example, genera within this group range from the strictanaerobes to the strict aerobes. Some of these organisms are importantmedical pathogens, while many are saprophytic organisms which benefitthe environment by degrading dead biological or organic matter. Whilemany of these organisms grow optimally at temperatures common in theenvironment (e.g., 25-27° C.), some organisms are quite capable ofgrowing at the body temperature of most mammals (e.g., approximately35-37° C.). However, two genera of medically important actinomycetes(Thermomonospora and Micropolyspora) are true thermophiles, capable ofgrowing at temperatures ranging to 50° C.

Colonies may be bacterium-like (i.e., ranging from butyrous to waxy andglabrous), or fungus-like (i.e., heaped, leathery, membranous coloniesthat are covered with aerial hyphae). Many actinomycetes exhibitfilamentous growth with mycelial colonies, and some actinomycetes causechronic subcutaneous granulomatous abscesses much like those caused byfungi. Because of these similarities, the actinomycetes werelong-regarded as fungi, rather than bacteria (see e.g., G. S. Kobayashi,"Actinomycetes: The fungus-like bacteria," in B. D. Davis et al.,Microbiology, 4th ed., J. B. Lippincott Co., New York, 1990), pp.665-671).

Despite their similarities with the fungi, the actinomycetes havetypical prokaryotic characteristics in terms of nucleoid and cell wallstructure, antimicrobial sensitivity, the absence of sterols, motilityby means of simple flagella, and long filaments of the diameter ofbacteria (approximately 1 μm, compared to the larger fungal hyphae).Microscopically, the morphology of the aerobic actinomycetes varieswidely between genera and species, although they are generally observedas grampositive rods or branching filaments. Some genera never progressbeyond a typical bacterium-like coccoid or bacillary form (e.g.,Rhodococcus sp.), while others form filaments with extensive branching(e.g., Nocardia, Streptomyces, Actinomadura, and Nocardiopsis). Most arenon-motile in their vegetative phase of growth. However, some generatend to form branched filaments which eventually fragment into motile,flagellated cells (e.g., Oerskovia sp.) (see e.g., G. Land et al.,."Aerobic pathogenic Actinomycetales," in A. Balows et al., Manual ofClinical Microbiology, 1991, pp. 340-359).

Most of the actinomycetes form spores, with the type of spore formationserving as a phylogenetic and taxonomic tool for separating theorganisms into groups. The actinomycetes are highly diverse, with atleast ten subgroups. They are also closely related to such organisms asthe coryneform group (e.g., Corynebacterium sp.), the propionic acidbacteria (e.g., Propionibacterium sp.), and various obligate anaerobes(e.g., Bifidobacterium, Acetobacterium, Butyrvibrio, andThermoanaerobacter). The following table lists the organisms included inthe suprageneric groups of actinomycetes as set forth in the most recentedition of Bergey's Manual, vol. 4, (Stanley T. Williams, editor of vol.4; John G. Holt, editor in chief, Bergey's Manual® of SystematicBacteriology, Williams & Wilkins, pp. 2334-2338 [1989]).

                  TABLE 1                                                         ______________________________________                                        Actinomycetes Groups                                                                                  Representative                                        ______________________________________                                          Number Group Groups/Genera                                                  ______________________________________                                        I     Actinobacteria                                                                              Group A: Agromyces,                                           Aureobacterium                                                                Group B: Arthrobacter, Rothia                                                 Group C: Cellulomonas, Oerskovia                                              Group D: Actinomyces,                                                         Arcanobacterium                                                               Group E: Arachnia, Pimelobacter                                               Group F: Brevibacterium                                                       Group G: Dermatophilus                                                      II Actinoplanetes Actinoplanes, Ampullariella,                                  Micromonospora                                                              III Maduromycetes Actinomadura pusilla group,                                   Microbispora, Streptosporangium                                             IV Micropolysporas Actinopolyspora, Faenia,                                     Saccharomonospora                                                           V Multilocular Sporangia Frankia, Geodermatophilus                            VI Nocardioforms Nocardia, Rhodococcus, Caseobacter                           VII Nocardioides Nocardiodes                                                  VIII Streptomycetes Streptomyces, Streptoverticillium,                          Kineosporia                                                                 IX Thermomonosporas Thermomonospora, Nocardiopsis,                              Actinomadura madurae group                                                  X Other Genera Glycomyces, Kitasatosporia                                       Spirillospora, Thermoactinomyces                                          ______________________________________                                    

Although these organisms may often be identified to the genus levelbased on their morphology at the time of primary isolation, organismsthat have been repeatedly transferred in the laboratory often do notretain their typical morphologic characteristics and must be identifiedbiochemically, or by analysis of their membrane fatty acid composition.Serological methods for identification and differentiation are rarelyused, due to the extensive degree of cross-reactivity among theactinomycetes (see e.g ., G. S. Kobayashi, supra, at p. 666).

II. Importance of the Actinomycetes as Pathogens

Many of these organisms are soil-dwellers, with relatively littlepathogenic capabilities. Indeed, the actinomycetes are among the mostabundant of organisms in the soil, where they serve the importantfunction of breaking down proteins, cellulose, and other organic matter.Nonetheless, some Actinomyces, Nocardia, and Streptomyces species areassociated with diseases of medical and veterinary importance,especially in immunocompromised individuals. The spectrum of diseasecaused by the actinomycetes is extremely broad, with pathology that isdependent upon a combination of organism type, tissue involved, and theimmune status of the host. In immunocompetent humans, the most commondiseases are a non-invasive, acute or chronic allergic respiratorysyndrome (e.g., farmer's lung), and mycetoma. In immunocompromisedindividuals, infection often begins in the lung as an acute to chronicsuppurative process, which may progress to cavitation and multi-lobularpulmonary disease. In these patients, infection may spread to otherorgan systems. Importantly, these organisms have a predilection for thecentral nervous system.

Several species of Actinomyces have been associated with actinomycosisin humans and other animals, with A. israelii being the most commonhuman isolate, and A. bovis the most common cattle isolate.Actinomycosis is usually characterized by chronic, destructive abscessesof connective tissues. Abscesses expand into the neighboring tissues andeventually produce burrowing, tortuous sinus tracts to the surface ofthe skin, where purulent material is discharged. In cattle, the lesionsare characteristically large abscesses of the lower jaw (hence thecommon name of the disease, "lumpy jaw"), usually with extensive bonedestruction. As with most saprophytic organisms that occasionally causedisease, actinomycosis is not transmissible from person to person, norbetween humans and other animals. Indeed, it is difficult to establishinfection in laboratory animals.

For in vitro growth in the laboratory, these pathogenic organisms tendto be microaerophilic (e.g., require a decreased oxygen tension foroptimum growth), require rich growth media, optimum incubationtemperatures of 37° C., and about 7 days of incubation. Althoughactinomycetes are soil organisms, actinomycosis is usually caused byendogenous organisms that have colonized the individual, rather thanorganisms from the environment. The organism is usually a commensal,which can be cultured from the tonsils of most humans, and is almostalways present in teeth and gum scrapings. The conditions that lead toinvasiveness are not well characterized, but may be multi-factorial, asactinomycotic infections are often mixed, with various organisms (e.g.,Haemophilus actinomycetemcomitans, Eikenella corrodens, Fusobacterium,and Bacteroides) also present.

In contrast to the Actinomyces, diseases due to Nocardia sp. areassociated with infection of the individual with soil organisms, ratherthan endogenous commensals. Nocardia are among the most clinicallyimportant actinomycetes, as they are responsible for the majority ofdisease associated with this group of organisms. Indeed, the term"nocardiosis" is often used synonymously for pulmonary and disseminatedinfection caused by any of the aerobic actinomycetes (see e.g., G. Landet al., "Aerobic Pathogenic Actinomycetales," in A. Balows et al.,Manual of Clinical Microbiology, 5th ed., American Society forMicrobiology, Washington, D.C., 1991, pages 340-359).

There are two common forms of disease associated with Nocardia sp.,namely, pulmonary nocardiosis resulting from inhalation of the organism,and mycetoma, which is characterized by chronic subcutaneous abscessesresulting from contamination of skin wounds. These infections areusually serious, especially as they are frequently seen in associationwith immunosuppression or chronic underlying diseases (e.g., carcinoma,chronic granulomatous disease, Hodgkin's disease, and leukemia). Onceclinically evident, the progression of nocardiosis tends to beprogressive and fatal, with approximately 50% of patients dying, evenwith aggressive therapy (see e.g., G. S. Kobayashi, "Actinomycetes: TheFungus-Like Bacteria, in B. D. Davis et al. (eds.), Microbiology, 4thed., J. B. Lippincott Co., Philadelphia [1990], pages 665-671).

The Nocardia are aerobic organisms which grow on relatively simple mediaover a wide temperature range. As with the mycobacteria, growth inliquid media usually results in the production of a dry, waxy pellicleon the surface of the media. The two species most commonly associatedwith human disease, N. brasiliensis and N. asteroides, share many othercharacteristics with the mycobacteria. For example, they are somewhatacid-fast, more easily stained with fuchsin, and their cell wallscontain components characteristic of mycobacteria and corynebacteria(e.g., mycolic acid residues). Unlike the great majority of organisms,the somewhat harsh methods used to isolate mycobacteria (e.g., treatmentof samples with N-acetyl-L-cysteine, and sodium hydroxide) are oftensuccessful for isolation of Nocardia. Extensive serologiccross-reactions in agglutination and complement fixation tests furtherindicate the relatedness of these groups of organisms.

The Streptomyces are also sometimes associated with actinomycoticabscesses. Mycetomas caused by streptomycetes are clinicallyindistinguishable from those caused by other actinomycetes. However,identification of these organisms can be critical, as they are generallynot susceptible to antimicrobial agents. Therefore, treatment oftenentails surgical removal of the affected area or amputation.

Other members of the actinomycetes are capable of causing disease,including allergic respiratory disease ("farmer's lung"), which occursin agricultural workers who inhale dust from moldy plant material. Thissyndrome has been associated with at least three thermophilicactinomycetes (Thermopolyspora polyspora, Micromonospora vulgaris, andMicropolyspora faeni). This disease is very similar to that caused byinhalation of allergens produced by various fungi, particularlyAspergillus sp.

In addition to the pathogenic potential of this group of organisms,there is also great interest in the particular genera which produceantimicrobial compounds.

III. Industrial Importance of the Actinomycetes

Ever since Waksman isolated actinomycin in 1940, and streptomycin in1943, the streptomycetes have attracted a large amount of attention (seee.g., G. S. Kobayashi, et al., at p. 671). Thousands of soil samplescollected world-wide have resulted in the identification of over 90% ofthe therapeutically useful antibiotics (see e.g., G. S. Kobayashi,"Actinomycetes: The Fungus-Like Bacteria, in B. D. Davis et al (eds.),Microbiology 4th ed., J. B. Lippincott Co., Philadelphia [1990], pages665-671). The interest in improving antibiotic qualities and yields hasresulted in various studies on this group of organisms, includingimproved methods for their growth and characterization.

It is important that strains be differentiated in screening programs toidentify antibiotic activities, so that redundant testing is avoided. Inaddition, differentiation facilitates determination of taxonomicrelationships which may lead to other organisms with promisingactivities. Unfortunately, testing of these organisms is often verydifficult. Because they grow as filaments, they have a strong tendencyto form clumps of mycelia which makes them much more difficult tohandle, both in liquid cultures and on solid or semi-solid agar media.Furthermore, because of their complex life cycle which involvessporulation and germination, it is very difficult to obtain cultureswhich perform consistently in metabolic and biochemical testingprograms. In addition, the presence of spores and the potential fortheir inhalation, represents a safety hazard to personnel responsiblefor the cultivation and characterization of these organisms, especiallyin settings where large-scale growth is necessary (e.g., antimicrobialproduction).

These growth characteristics also contribute to the difficultiesassociated with determining the susceptibility of the actinomycetes toantimicrobial compounds. The most frequently used testing methods are amodified Kirby-Bauer disk diffusion method agar dilution, and a minimalinhibitory concentration (MIC) method (see e.g., G. Land et al.,"Aerobic Pathogenic Actinomycetales," in A. Balows et al., Manual ofClinical Microbiology, 5th ed., American Society for Microbiology,Washington, D.C., [1991], at p. 356). However, the success of thesemethods is contingent upon the production of a homogenized suspensionfor use as a standardized inoculum. Most commonly, agitation withsterile glass beads or a tissue homogenizer is used to prepare ahomogenous suspension that can then be diluted to a 0.5 McFarlandstandard prior to inoculating the test media (see e.g., G. Land et al.,"Aerobic Pathogenic Actinomycetales," in A. Balows et al., Manual ofClinical Microbiology, 5th ed., American Society for Microbiology,Washington, D.C., 1991, pages 340-359). These methods involving physicalhomogenization are very labor-intensive and tedious, and they result indamage, fragmentation, and death of some fraction of the cells.Furthermore, the additional manipulation required to produce ahomogenous suspension prior to inoculation increases the risk ofcontamination of laboratory personnel and the laboratory environment.

Therefore, what is needed is a safe, reliable, easy-to-use system forthe characterization and testing of medically and industrially importantorganisms, including but not limited to organisms such as theactinomycetes. In particular what is need is a rapid method that isreadily automatable and useful in various settings (e.g., clinical,veterinary and environmental laboratories, and industry). Methods andcompositions are also needed for high-volume, reliable analysis ofstrain differences between organisms.

SUMMARY OF THE INVENTION

The present invention relates to growing and testing any cell type in amultitest format which utilizes a gel forming matrix for the rapidscreening of clinical and environmental cultures. In particular, thepresent invention is suited for the characterization of commonlyencountered microorganisms (e.g., E. coli, S. aureus, etc.), as well ascommercially and industrially important organisms from various anddiverse environments. For example, the present invention is particularlysuited for the growth and characterization of bacteria, as well as theactinomycetes and fungi (e.g., yeasts and molds).

In one embodiment, the present invention provides methods for testingmicroorganisms comprising the steps of: providing a testing meanscomprising redox purple and one or more test substrates; introducingmicroorganisms into the testing means; and detecting the response of themicroorganism to the one or more test substrates. In a preferredembodiment, the testing substrates are selected from the groupconsisting of carbon sources and antimicrobials.

In an alternate embodiment, the testing means further comprises one ormore gel-initiating agents. In a preferred embodiment, thegel-initiating agent comprises cationic salts. In another alternativeembodiment, the testing means further comprises one or more gellingagents. In a preferred embodiment, the microorganisms are in an aqueoussuspension. In another preferred embodiment, the aqueous suspensionfurther comprises one or more gelling agents. It is contemplated thatvarious gelling agents will be used with the present invention,including, but not limited to agar, Gelrite™, carrageenan, and alginicacid.

In one embodiment of the method, the microorganisms are bacteria, whilein another embodiment, the microorganisms are fungi. It is alsocontemplated that the methods of the present invention will be used withmembers of the Order Actinomycetales.

It is contemplated that various testing means will be used in thepresent invention. In one preferred embodiment, the testing meanscomprises at least one microplate, in an alternative embodiment, thetesting means comprises at least one microcard. In yet anotherembodiment, the testing means comprises at least one petri plate.

The present invention also provides a kit, comprising redox purple andone or more test substrates. In a preferred embodiment, the testsubstrates are selected from the group consisting of carbon sources andantimicrobials. In another preferred embodiment, the kit furthercomprises one or more gel-initiating agents. In a particularly preferredembodiment, the gel initiating agent comprises cationic salts. In analternative preferred embodiment, the kit further comprises one or moregelling agents. In another preferred embodiment, the gelling agent isselected from the group consisting of agar, Gelrite™, carrageenan, andalginic acid.

In another embodiment, the kit further comprises a suspension ofmicroorganisms. In one preferred embodiment, the kit further comprises atesting means. It is contemplated that various testing means fornatswill be used successfully in various embodiments of the kits of thepresent invention, including microplates, microcards, petri plates, andany other suitable support in which the testing reaction can occur.

In yet another embodiment, the present invention provides a kit,comprising redox purple and one or more gelling agents. It iscontemplated that various gelling agents will be used successfully inthe various embodiments of the kits of the present invention, includingbut not limited to agar, Gelrite™, carrageenan, and alginic acid. In onepreferred embodiment, the kit further comprises one or moregel-initiating agents. In a particularly preferred embodiment, thegel-initiating agent comprises cationic salts. In an alternativeembodiment, the kit further comprises a suspension of microorganisms.

In an alternative embodiment, the kit further comprises one or more testsubstrates. It is contemplated that the test substrates included in thekit of the present invention be selected from the group consisting ofcarbon sources and antimicrobials.

In yet another embodiment, the kit further comprises a testing means. Itis contemplated that various testing means formats will be usedsuccessfully in various embodiments of the kits of the presentinvention, including microplates, microcards, petri plates, and anyother suitable support in which the testing reaction can occur.

The present invention describes test media and methods for the growth,isolation, and presumptive identification of microbial organisms. Thepresent invention contemplates compounds and formulations, as well asmethods particularly suited for the detection and presumptiveidentification of various diverse organisms.

In order to characterize or identify organisms present in a sample, thepresent invention combines a gel-forming suspension with microorganismsthat are already in the form of a pure culture. This is in contrast tothe traditional pour plate method which involves heated agar and asample that contains a mixed culture (see e.g., J. G. Black,Microbiology: Principles and Applications, 2d ed., Prentice Hall,Englewood Cliffs, N.J., p. 153 [1993]; and American Public HealthAssociation, Standard Methods for the Examination of Water andWastewater, 16th ed., APHA, Washington, D.C., pp. 864-866 [1985]).

It is also in contrast to the pour plate method of Roth (U.S. Pat. Nos.4,241,186, and 4,282,317), which utilizes a solidifying pectinsubstance. In the present invention, colloidal gel-forming substancesare used at low concentrations, forming soft gels or viscous colloidalsuspensions that do not need to, and in fact work best, when notcompletely solidified into a rigid gel.

In one embodiment, the present invention provides a method forintroducing microorganisms into a testing device, comprising the stepsof providing a testing device comprising a plurality of testing wells orcompartments, wherein each compartment contains one or moregel-initiating agents; preparing a suspension comprising a pure cultureof microorganisms and an aqueous solution containing a gelling agent,under conditions such that the suspension remains ungelled; andintroducing the suspension into the testing device under conditions suchthat the suspension contacts the gel-initiating agents present in thecompartments and results in the production of a gel or colloidal matrix.

In another embodiment, the present invention provides a method fortesting microorganisms comprising the steps of providing a testingdevice comprising a plurality of testing compartments, wherein thecompartments contain a testing substrate and one or more gel-initiatingagents; preparing a suspension comprising a pure culture ofmicroorganisms and an aqueous solution comprising a gelling agent underconditions such that the suspension remains ungelled; introducing thesuspension into the compartments of the testing device under conditionssuch that the suspension forms a gel matrix within the compartment; anddetecting the response of the microorganisms to the testing substrate.In one preferred embodiment, the testing device is a microplate.

It is contemplated that the microorganisms tested in this method will bebacteria, including members of the Order Actinomycetales, or fungi(e.g., yeasts and molds).

In one embodiment, the gelling agent is selected from the groupconsisting of Gelrite™, carrageenan, and alginic acid. In a particularlypreferred embodiment, the gelling agent is carrageenan which containspredominantly iota-carrageenan. In one embodiment, the gel-initiatingagent comprises cationic salts.

In one embodiment, the testing substrates are selected from the groupconsisting of carbon sources and antimicrobials. In yet anotherembodiment, the method further includes a colorimetric indicator,wherein the colorimetric indicator is selected from the group consistingof chromogenic substrates, oxidation-reduction indicators, and pHindicators.

In yet another embodiment, the present invention encompasses a kit forgrowth and identification of microorganisms comprising: a testing devicecomprising a plurality of testing compartments containing one or moregel-initiating agents; and an aqueous solution comprising a gellingagent. In one preferred embodiment, the testing compartments furthercontain testing substrates, such as carbon sources and antimicrobials.In one embodiment, the gel-initiating agent comprises cationic salts.

In one embodiment of this kit, the testing device is a microplate. In apreferred embodiment, the kit contains a gelling agent that is selectedfrom the group consisting of Gelrite™, carrageenan, and alginic acid. Inone preferred embodiment, the gelling agent is a carrageenan whichpredominantly contains the iota form of carrageenan. In one embodiment,the gel-initiating agent comprises cationic salts.

It is contemplated that the kit of the present invention will be usedwith microorganisms such as bacteria, including members of the OrderActinomycetales, as well as fungi (e.g., yeasts and molds).

It is also contemplated that the kit will also include a colorimetricindicator selected from the group consisting of chromogenic substrates,oxidation-reduction indicators, and pH indicators.

In an alternative embodiment, the present invention comprises a kit forcharacterizing and identifying microorganisms comprising: a microplatetesting device containing a plurality of compartments, wherein thecompartments contain one or more gel-initiating agents and one or moretesting substrates, wherein the testing substrates are selected from thegroup consisting of antimicrobials and carbon sources and an aqueoussuspension comprising a gelling agent.

In one embodiment of this kit, the testing device is a microplate. In apreferred embodiment, the kit contains a gelling agent that is selectedfrom the group consisting of Gelrite™, carrageenan, and alginic acid. Inone preferred embodiment, the gelling agent is a carrageenan whichpredominantly contains the iota form of carrageenan. In one embodiment,the gel-initiating agent comprises cationic salts.

It is contemplated that the kit of the present invention will be usedwith microorganisms such as bacteria, including members of the OrderActinomycetales, as well as flngi (e.g., yeasts and molds).

It is also contemplated that the kit will include a calorimetricindicator selected from the group consisting of chromogenic substrates,oxidation-reduction indicators, and pH indicators.

The present invention also provides methods for comparing the functionof a gene in at least two cell preparations, comprising the steps of:providing a testing device comprising a plurality of testing wells,wherein the wells contain a testing substrate and one or moregel-initiating agents; preparing a first suspension comprising a firstcell preparation, in an aqueous solution comprising a gelling agent, anda second suspension comprising a second cell preparation, in an aqueoussolution comprising a gelling agent, under conditions such that thefirst and second suspensions remain ungelled; introducing the first andsecond suspension into the wells of the testing device under conditionssuch that the first and second suspensions form a gel matrix within thewells, such that the first and second cell preparations are within thegel matrix; detecting the response of the first and second cellpreparations to the testing substrate; and comparing the response of thefirst and second cell preparations. In some embodiments, the first andsecond cell preparations comprise microorganisms selected from the groupconsisting of bacteria and f ungi. In yet other embodiments, the firstand second cell preparations contain cells of the same genus andspecies, while in still other embodiments, the firstthat differ in oneeparations contain cells that differ in one or more genes.

In alternative embodiments of the methods, the gelling agent is selectedfrom the group consisting of Gelrite™, carrageenan, and alginic acid. Infurther embodiments, the testing substrates are selected from the groupconsisting of carbon sources, nitrogen sources, sulfur sources,phosphorus sources, amino peptidase substrates, carboxy peptidasesubstrates, oxidizing agents, reducing agents, mutagens, amino acidanalogs, sugar analogs, nucleoside analogs, base analogs, dyes,detergents, toxic metals, inorganics, and antimicrobials. In still otherembodiments, the gel-initiating agent comprises cationic salts. In somepreferred embodiments, the methods further comprise a colorimetricindicator. In particularly preferred embodiments of the methods, thecolorimetric indicator is selected from the group consisting ofchromogenic substrates, oxidation-reduction indicators, and pHindicators. In some particularly preferred embodiments, theoxidation-reduction indicator is tetrazolium violet, while in otherembodiments, the oxidation-reduction indicator is redox purple. In yetother preferred embodiments, the testing device is at least onemicroplate. In further preferred embodiments, the response is a kineticresponse.

The present invention also provides kits suitable for determining thephenotype of at least two organisms, comprising: a microplate testingdevice containing a plurality of wells, wherein the wells contain one ormore gel-initiating agents and one or more testing substrates; a firstaqueous suspension comprising a gelling agent; and a second aqueoussuspension comprising a gelling agent.

In one preferred embodiment of the kits, the testing substrates areselected from the group consisting of carbon sources, nitrogen sources,sulfur sources, phosphorus sources, amino peptidase substrates, carboxypeptidase substrates, oxidizing agents, reducing agents, mutagens, aminoacid analogs, sugar analogs, nucleoside analogs, base analogs, dyes,detergents, toxic metals, inorganics, and antimicrobials. In alternativepreferred embodiments of the kits, the gelling agent is selected fromthe group consisting of Gelrite™, carrageenan, and alginic acid. Instill other embodiments of the kit, the gel initiating agent comprisescationic salts. In some particularly preferred embodiments, the testingdevice f urther comprises a colorimetric indicator selected from thegroup consisting of chromogenic substrates, oxidation-reductionindicators, and pH indicators. In alternate preferred embodiments, theoxidation-reduction indicator is tetrazolium violet, while in otherembodiments, the oxidation-reduction indicator is redox purple.

The present invention further provides methods and compositions forextrapolating the finctions of genes or genetic sequences in variouscell types. For example, the present invention provides methods forextrapolating the function of genes or genetic sequences in eukaryoticcells. In some embodiments, microbial genomes are examined to identifysequences that are homologous to the gene(s) or genetic sequence(s) ofinterest in the eukaryotic cell. Then, mutations are introduced into thehomologous microbial gene. Next, the phenotypes of the wild-type andmutant microbial cells are analyzed and/or compared, as desired. Inother embodiments, the functions of the microbial and eukaryotic genesare compared by utilizing genetic engineering methods to preparetransferable expression vectors (e.g., plasmids, phages, etc.)containing the eukaryotic gene(s) or genetic sequence(s) of interest.This expression vector is transferred into and expressed in a microbialhost cell. The phenotype of the host microbial cell (i.e., the cellcontaining the expression vector) and untransformed microbial cells(i.e., control cells comprising the same microbial cell line, but notcontaining the expression vector) are then analyzed and/or compared, asdesired. In further embodiments, the vector comprises eukaryotic genesthat have been modified (ie., the genes are modified such that they arenot the same as the wild type gene sequences).

DESCRIPTION OF THE FIGURES

FIG. 1 is an exploded perspective view of one embodiment of the deviceof the present invention.

FIG. 2 is a top plan view of the device shown in FIG. 1.

FIG. 3 is a cross-sectional view of the device shown in FIG. 2 along thelines of 3--3.

FIG. 4 is a bottom plan view of the device shown in FIG. 1.

FIG. 5 shows the synthesis pathway of redox purple.

GENERAL DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that variouscells (e.g., microbial strains) can be differentiated based ondifferential biochemical reactions. Surprisingly, it was determinedduring the development of the present invention that the biochemicalreactions work best when the cells are contained within a gel matrix.The present invention incorporates a multiple test format in a testingdevice, for presumptive and rapid microbiological screening of variousclinical, veterinary, research, industrial and environmental specimens.It is also intended that the present invention will be useful fordefinitive identification and diagnosis. In preferred embodiments, thepresent invention is suitable for the comparative phenotype testing ofmicroorganisms and other cells. It is intended that comparativephenotypic testing will find use in functional genomics (ie., wherebycells and/or microbial strains that differ in a defined set of genetictraits are compared). It is not intended that the invention be limitedto a particular genus, species nor group of organisms. Indeed, it isalso intended that the present invention will find use with cells of anytype, including, but not limited to cells maintained in cell culture,cell lines, etc., including mammalian, plant, and insect cells. Thecompositions and methods of the present invention are particularlytargeted toward some of the most economically important organisms, aswell as species of clinical importance.

The present invention contemplates an indicator plate essentiallysimilar in structure to microtiter plates ("microplates" or"MicroPlates™") which are commonly used in the art and commerciallyavailable from numerous scientific supply sources (e.g., Biolog, Fisher,etc.). It is contemplated that the present invention be used withvarious gelling agents, including but not limited to alginate,carrageenan, and gellan gum (e.g., Gelrite™; Phytagel™).

Because the cells are trapped within the gel matrix, the presentinvention is a great improvement over standard microplate testingmethods in which liquid cultures are used. Unlike the liquid format, thegel matrix of the present invention does not spill from the microplate,even if the plate is completely inverted. This safety considerationhighlights the suitability of the present invention for use withorganisms or other cells that are easily aerosolized. It is alsocontemplated that the present invention is highly useful in theeducational setting, where safety is a primary concern. The presentinvention permits novices to work with bacteria and study theirbiochemical characteristics with a reduced chance of contamination, ascompared to other testing systems. In addition, the present inventionpermits novices to work with infected cells (e.g., virally-infectedcells harvested from cell cultures), with a reduced chance ofcontamination.

The gel matrix system of the present invention also offers otherimportant advantages. For example, over incubation periods of severalhours, cells will often sink to the bottom of testing wells and/orattach or clump to other cells, resulting in a non-uniform suspension ofcells within the wells. This non-uniformity can result in a non-uniformresponse of the cells in the well. Clumping artifacts perturb theoptical detection of cellular responses. Thus, because the presentinvention provides methods and compositions which trap the cells in agel matrix within the wells, the cells are uniformly suspended, and haveuniform access to nutrients and other compounds in the wells. Thus, thepresent invention serves to make this type of cell testing asreproducible and homogenous as possible. Furthermore, in naturalsettings, cells often grow attached to surfaces or in contact with othercells (e.g., in biofilms or monolayers). By providing contact betweenthe cells and a semi-solid, gel support, the gel matrix of the presentinvention simulates the natural state of cell growth. In addition, thegel matrix decreases the diffusion of oxygen to the cells and helpsprotect them from oxidative damage.

As various cells may be characterized using the present invention, it isnot intended that the choice of primary isolation or culture media belimited to particular formulae. In addition to commonly isolatedorganisms, the range of cell types that can be tested using the methodsand compositions of the present invention includes cells that undergocomplex forms of differentiation filamentation, sporulation, etc. Forexample, in one embodiment, organisms such as the actinomycetes aregrown on an agar medium which stimulates the production of aerialconidia. This greatly facilitates the harvesting of organisms forinoculation in the present invention. However, it is not intended thatthe present invention be limited to actinomycetes. Indeed, the presentinvention provides methods and compositions for the testing of fungi(e.g., yeasts and molds), as well as bacteria other than actinomycetes.As with the actinomycetes, these organisms may be grown on any primaryisolation or culture medium that is suitable for their growth, althoughit is preferred that the primary isolation or culture medium usedpromotes the optimal growth of the organisms. For cell lines and cellcultures (i.e., mammalian, plant, and/or insect cells maintained invitro), the cells are grown in cell culture media (e.g., Eagle's MinimalEssential Medium, etc.), suitable for the cell growth.

In one embodiment, a microtiter (e.g., microplate) format is used. Inthis embodiment, the gel-forming matrix containing suspended cells isused to inoculate the wells of a microtiter plate or another receptacle.At the time of inoculation, the gel-forming matrix is in liquid form,allowing for easy dispensing of the suspension into the compartments.These compartments contain dried biochemicals and cations. Upon contactof the gel-forming matrix with the cations, the suspension solidifies toform a soft gel, with the cells evenly distributed throughout. This gelis sufficiently viscous or rigid that it will not fall out of themicrotiter plate should the plate be inverted.

In another embodiment, a microcard format is used. As shown in FIGS.1-4, one embodiment of the device of the present invention comprises ahousing (100) with a liquid entry port through which the sample isintroduced. The housing further contains a channel (110) providingcommunication to a testing region (120) so that a liquid (not shown) canflow into a plurality of wells or compartments (130). The channel (110)is enclosed by the surface of a hydrophobic, gas-venting membrane (140)adapted for forming one surface of the wells (130) and attached to oneside of the housing (100). The housing (100) can be sealed on its otherside by a solid base (150). In other embodiments, a flexible tape (notshown) may be substituted for the solid base (150) or the solid base(150) may be molded so as to be integral with the housing (100).

After filling the device with the gel-forming matrix containing cells,(not shown) an optional non-venting material such as tape (e.g.,polyester tape) (160) can be adhered to the outer surface of thegas-venting membrane (140) to seal it against evaporation of the gelmatrix within the device through the gas-venting membrane. At the timeof delivery, the gel-forming matrix with suspended cells is in liquidform. Once the liquid comes into contact with the compounds present inthe testing region, a gel matrix is produced, trapping the suspendedcells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the discovery thatvarious cells or cell types may be identified and differentiated basedon differential biochemical reactions observed in gelled media. Themultiple test medium of the present invention permits presumptive andrapid microbiological screening of various specimens. In particular,this invention in the form of a kit, is suitable for the easy and rapidbiochemical testing of various cells, including commonly isolatedbacteria, as well as actinomycetes and fungi (i.e., yeasts and molds),in addition to mammalian, insect, and plant cells. In particular, thepresent invention provides compositions and methods for the phenotypicanalysis of cells.

Phenotypic Analysis

The Darwinian belief in a common ancestry of Earth's gene pool and theconcept of evolution by gene duplication, mutation, and rearrangementare at the foundation of the new field of genomics, a field that hasevolved rapidly in recent years by successfully utilizing microorganismsas models. In genomic analysis, genes whose function(s) and codedprotein are known in one cell type are used as a basis for extrapolationwhen a similar coding sequence is found in another cell type.

Initially, the pace of genomic research was limited by DNA sequencingtechnology. However, with new techniques developed in recent years, thepace of genomic sequencing has greatly accelerated and the sequencingeffort is no longer considered a rate limiting step. Although thecomplete sequence of the human genome (approx. 75,000 genes) is stillseveral years away, great strides have been made in the sequencing ofsingle-celled microorganisms, which have smaller genomes (approx. 470genes in the bacterium Mycoplasma genitalium to approx. 12,000 genes inthe protozoan Oxytricha similis). As of September, 1997, the completegenomic sequences of 12 microbes had been obtained (See, Pennisi,Science 277: 1432-1434 [1997]), representing the three domains ofcellular life: eubacteria (e.g., Escherichia coli, and Bacillussubtilis), archaea (e.g., Methanococcus jannaschii, and Methanobacteriumthermoautotrophicum), and eucarya (e.g., Saccharomyces cerevisiae). Theannotation of genes corresponding to open reading frames (ORFs) reliesheavily on microorganisms, especially E. coli. Often the extrapolationfrom DNA sequence to enzyme or regulatory function is based uponsequence data from the best studied microbes (e.g., E. coli, B.subtilis, and S. cerevisiae) or from heterologous sequences that arecloned into E. coli. Yet even with a great deal of extrapolation, thepercentage of genes with an "ascribed function" ranges from only 44% to69%. There is a tremendous amount of functional information that remainsto be determined and understood. Indeed, genome sequencing has reached aturning point, as indicated by Smith et al., "The next importantchallenge is to determine, in an efficient and reliable way, somethingabout the function of each gene in the genomes" (Smith et al., Science274: 2069-2074 [1997]).

Over the past three decades, biologists have sought tools that wouldallow them to understand the workings of cells by analyzing all of thecell's genes simultaneously. The first breakthrough in this endeavor of"global analysis" came in the early 1970's with the introduction of onedimensional protein electrophoresis, which allowed scientists toseparate and observe nearly all of a cell's proteins. This innovationwas soon followed by the superior resolution obtained by two dimensionalseparations. One dimensional methods were next developed for DNA andmRNA analysis (i.e., Southern and Northern blot analysis).

Nucleic acid microarrays (See e.g., DeRisi et al., Science 278: 680-686[1997]) and gene fusion arrays (See e.g., Glaser, Genet. Enginer. News,Sep. 15, 1997, at pages 1 and 15), have been developed which can analyzethe genotype and gene expression levels of cells.

By determining the function of genes, the analysis can go a stepfurther, through the ascertainment of groups of genes which areregulated similarly and which, by implication, are likely to providerelated functions in the cell. Though clearly of great value, thesetechnologies still do not indicate the function of the gene, nor do theydescribe the phenotypic changes that occur in the cell of interest dueto the presence of different alleles of that gene. The present inventionsolves these problems, by providing methods and compositions to assaythe function of genes directly in cells. Unlike previous methods andcompositions, the present invention permits the analysis of thousands ofcell phenotypes simultaneously. This cellular approach is nicelycomplementary to the molecular techniques; it is contemplated that thoseskilled in the art will utilize the present invention in conjunctionwith molecular methods to characterize a wide variety of cell types.

Phenotypic Analysis of Yeast

As indicated above, the present invention is intended for use witheukaryotic, as well as prokaryotic cells. Indeed, the ease of findingphenotypic changes has also been demonstrated recently in yeast. As of1996, of the 6000 genes in the chromosome of S. cerevisiae, less thanone half had been known, and 30% could not be assigned a function(Goffeau et al., Science 274: 546-567 [1996]). Subsequently, Smith andcoworkers developed a method that allowed the introduction of Tylinsertion mutations into 97% of the genes on chromosome V. Testing thiscollection with only seven phenotypic tests based on the growth rate ofthe organism on certain media, they found detectable changes in 61.6% ofthe mutant strains (Smith et al., Science 274: 2069-2074 [1996]).Moreover, these authors observed that disruption of many genes resultedin multiple phenotypes, and in fact uncovered previously undetectedphenotypes for previously described genes, some of which were quiteunexpected. In contrast, the present invention provides a much largernumber, as well as more narrow phenotypic tests that provide much moredetailed information about the change(s) in cell physiology that aredetected in the yeast cells.

Summary of the Methods

The present invention provides useful, practical, efficient andcost-effective systems, including in one embodiment, an instrument whichis used in conjunction with disposable testing panels, to allow thedirect and simultaneous analysis of cells and cell lines for thousandsof phenotypes. The present invention provides methods and compositionsfor the phenotypic analysis of prokaryotic, as well as eukaryotic cells.Indeed, the present invention is not limited to any particular organism,cell, or testing format.

In many embodiments, the present invention provides one or more testingpanels, with each test panel including substrates for 95 phenotypictests. In one embodiment, the substrates in the test panel includevarious carbon sources, while in other embodiments, the test panelsinclude nitrogen, sulfur, phosphorus, and/or other substrates. Thus, itis intended that the present invention encompass testing panels withtest substrates of any type suitable for the phenotypic testing ofvarious cells.

In one preferred method, the present invention encompasses methods andcompositions for the phenotypic testing of E. coli, which is animportant "model" organism for many biochemical systems. In anotherembodiment, the present invention provides methods and compositions forthe testing of isogenic strains with known mutations, in order toidentify and characterize unexpected and/or misleading phenotypes.

In other preferred embodiments, the present invention provides methodsand compositions to determine the function of genes of interest. Forexample, the present invention provides means to analyze and comparesource strains and daughter strains for their phenotypic differences.Thus, in one embodiment, the gene of interest, with an unknown functionin the source strain, is completely or partially inactivated by creatingan altered allele in an isogenic daughter strain. Then, the sourcestrain and the daughter strains are cultured simultaneously underidentical conditions and tested in the testing panels described above inorder to determine the phenotypic consequences of the alteration of genefunction.

In other embodiments, a third cell strain is created. This third strainis a revertant of the mutation, derived from the daughter strain. It isintended that this approach will find use in situations in which thecells contain mutations that strongly select for secondary suppressormutations in the cell line that otherwise can easily go unnoticed. Byanalyzing a revertant along with the source and daughter strains, onecan tell whether any and all phenotypic differences between source anddaughter are due to the original mutation or to second site mutations.

In still other embodiments, a gene of interest from another cell type issequenced and its homolog is mutated in E. coli and/or S. cerevisiae. Inyet other embodiments, a gene of interest from another cell type iscloned and expressed at a physiologically appropriate level in E. coliand/or S. cerevisiae. In addition, the present invention providesmethods and compositions for the direct phenotypic analysis of cellswhich have been mutated. The present invention further contemplatesknocking out expression of genes transiently with antisense RNA, andperforming phenotypic analysis on cells with a transiently inactivatedgene.

One limitation of the current phenotypic testing methods is the range ofphenotypic tests covered, which is currently limited to carbon sourceoxidation tests. In contrast, the present invention provides methods andcompositions for the analysis of thousands of phenotypiccharacteristics. For example, in some embodiments, one or more sets of95 tests will be aimed toward each of the following groups of tests,which encompasses the majority of the catabolic functions of cells, aswell as the majority of the biosynthetic functions of cells, and much ofthe macromolecular machinery of the cell including the ribosome, DNA andRNA polymerases, cellular respiration, transport and detoxificationsystems, cell wall, and inner and outer membranes: (1) carbon sourceoxidation tests (including peptide substrates), (2) carbon sourcefermentation tests, (3) amino and/or carboxy peptidase tests, (4)nitrogen source tests, (5) phosphorus source tests, and/or sulfur sourcetests; (6) auxotrophic tests for all essential metabolites such as aminoacids, vitamins, polyamines, fatty acids, and/or nucleosides; (7)sensitivity tests for antibiotics and antimicrobials; (8) sensitivitytests for amino acid analogs, sugar analogs, nucleoside and baseanalogs, and/or mutagens; (9) sensitivity tests for dyes, detergents,heavy metals, oxidizing and/or reducing agents; and (10) other tests ofgeneral physiological interest such as growth at different pHconcentrations, salt concentrations, utilization of different osmoticbalancers, and/or ability to traverse various diauxic "shift-downs." Thegeneral issues in designing each group of tests are discussed below.

In addition to the carbon sources in such commercially available testingpanels as the ES MicroPlate™, it is contemplated that any number ofadditional carbon sources of interest will be included in the presentinvention. For example, it is contemplated that peptides be included ascarbon sources, as during the development of the present invention, itwas observed that these carbon sources can provide very usefulphenotypic tests. For example, it has been determined that E. coli canuse D-and L- alanine, D- and L- serine, D- and L-threonine, D- andL-aspartate, L-asparagine, L-glutarnine, L-glutamate, and L-proline ascarbon sources. It is further contemplated that various chromogenicamino and carboxypeptidase substrates be used in the present invention.

Carbon source fermentation tests measure acid production from a varietyof sugars, and therefore they can provide phenotypic information that isdifferent from carbon source oxidation tests. These tests are performedusing a chromogenic pH indicator, including, but not limited to suchcompounds as bromthymol blue, bromcresol purple, and neutral red.

The present invention also provides methods and compositions to observeutilization of nitrogen, phosphorus, and sulfur sources, using anindicator system (e.g., tetrazolium reduction) to demonstrate substrateutilization. Various nitrogen sources are contemplated for use in thepresent invention, including, but not limited to D-alanine, L-alanine,L-arginine, D-asparagine, L-asparagine, D-aspartic acid, L-asparticacid, L-cysteine, L-cystine, D-glutamic acid, L-glutamic acid,L-glutamine, glycine, L-histidine, L-homoserine, D,L-B-hydroxy-glutamicacid, L-isoleucine, L-leucine, L-phenylalanine, L-proline, D-serine,L-serine, L-tryptophan, L-tyrosine, glutathione (as well as any peptidecontaining the above amino acids), adenosine, deoxyadenosine, cytosine,cytidine, deoxycytidine, D-glucosamine, D-galactosamine, D-mannosamine,N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamtine, methylamine, ethylamine, butylamine, isobutylamine, amylamine,ethanolamine, ethylenediamine, pentamethylenediamine,hexamethylenetriamine, phenylethylamine, histamine, piperidine, pyrrole,B-alanine, glycocol, acetylglycocol, phenylglycine-o-carbonic acid,hippuric acid, urocanic acid, α-aminovaleric acid, γ-aminovaleric acid,α-aminoisovaleric acid, γ-aminoisovaleric acid, α-aminocaproic acid,γ-aminocaprylic acid, acetamide, lactamide, glucuronamide, formamide,propionamide, methoxylamide, thio-acetamide, cyanate, urea, diethylurea,tetraethylurea, biuret, parabanic acid, alloxan, alloxantine, allantoin,uric acid, theobromine, guanine, and xanthine.

Various phosphorous sources are contemplated for use in the presentinvention, including, but not limited to pyrophosphate,trimetaphosphate, 2'-mononucleotides, 3'-mononucleotides,5'-mononucleotides, 2', 3'-cyclic nucleotides, 3', 5'-cyclicnucleotides, aryl-phosphates (e.g., p-nitrophenyl phosphate),phosphonates (e.g., aminoethyl phosphonate), sugar phosphates (e.g.,glucose-1-phosphate), acid phosphates (e.g., 2-phospho-glyceric acid),aldehyde phosphates (e.g., glyceraldehyde-3 phosphate), α-glycerolphosphate, β-glycerol phosphate, inositol phosphates (e.g., phyticacid), phosphite, hypophosphite, and thiophosphate.

Various sulfur sources are contemplated for use in the presentinvention, including, but not limited to sulfur, thiosulfate,thiophosphate, metabisulfite, dithionite, tetrathionate, polysufide,cysteine, cystine, cysteic acid, cysteamine, cysteine sulphinic acid,cystathionine, lanthionine, ethionine, methionine, N-acetyl-methionine,N-acetyl-cysteine, glycyl-methionine, glycyl-cysteine, glutathione,L-djenkolic acid, L-2-thiohistidine, S-methyl-cysteine,S-ethyl-cysteine, methionine sulfoxide, methionine sulfone, taurine,thiourea, and thioglycolate.

In addition, various amino and carboxy peptidases are contemplated foruse in the present invention, including, but not limited to dipeptidescontaining all natural L-amino acids on the amino terninal, and allnatural L-amino acids on the carboxy terminal, as well as suitablenon-protein occurring amino acids, such as pyroglutamate, ornithine,α-amino butyrate, D-amino acids, etc.

The present invention also provides methods and compositions forauxotrophic testing using a minimal medium supplemented with varioussingle nutrients. In one embodiment, the growth in the well where theorganism is capable of using the nutrient results in a color change viatetrazolium reduction. Thus, mutations that result in auxotrophy causethe strain to fail to grow in all wells except the one containing thenecessary nutrient. In some cases, the wells contain more than onenutrient, in order to allow analysis of genes that affect more than onebiosynthetic pathways (e.g., isoleucine+valine (ilv), arginine+uracil(car), and purine+pyrimidine+histidine+tryptophan+nicotinamide (prs)).Various compounds are contemplated for use in this embodiment of thepresent invention, including, but not limited to L-amino acids,D-glutamic acid, D-aspartic acid, D-alanine, vitamins, nucleosides,polyamines, and fatty acids. In an alternative embodiment, a "drop out"medium or substrate is used. In this system, a complex definedsupplement is used and one nutrient is missing in the substratedispensed in each well (i.e., the medium lacks one nutrient of thesubstrate complex).

It is contemplated that for some embodiments of the present inventionfor sensitivity testing, a minimal medium is used, while in other cases,an enriched, defined medium is preferable. As in other reactions, in oneembodiment, growth in the wells can result in a color change viatetrazolium reduction. For each toxic agent, the optimal concentrationfor use in testing for sensitivity/resistance is determined for the celltype to be tested. Various sensitivity tests are contemplated, includingtests utilizing compounds including, but not limited to oxidizingagents, reducing agents, mutagens, antibiotics, amino acid analogs,sugar analogs, nucleoside and base analogs, dyes, detergents, toxicmetals, and toxic organics.

The present invention also provides methods and compositions for testinggrowth at extremes of pH and salt, and the compensatory effect ofseveral compatible solutes. In addition, diauxic testing is performedwith a limiting amount of a favored nutrient present in a well. In thisembodiment, the cells need to adapt from a more favored to a lessfavored nutrient, and the lag and growth kinetics for numeroussubstrates can be measured quickly and efficiently in a microtiter plateformat.

Indicator Plates of the Present Invention

The present invention also contemplates a multitest indicator plate thatis generally useful in the phenotypic characterization, as well asidentification and antimicrobial sensitivity testing of microorganisms.This medium and method are particularly targeted toward some of the mosteconomically important organisms, as well as species of clinicalimportance. It is not intended that the invention be limited to aparticular genus, species nor group of organisms. Indeed, it iscontemplated that any cell type (e.g ., microorganisms, as well asplant, mammalian, and insect cells) will find use in the presentinvention.

It is contemplated that the present invention be used with variousgelling agents, including, but not limited to agar, pectin, carrageenan,alginate, alginic acid, silica, gellans and gum. In one embodiment, thepectin medium of Roth (U.S. Pat. Nos. 4,241,186, and 4,282,317; hereinincorporated by reference) is used. However, this is not a preferredembodiment, as pectin is not a colorless compound itself. In oneparticularly preferred embodiment, the gellan of Kang et al. (U.S. Pat.Nos. 4,326,052 and 4,326,053, herein incorporated by reference) is used.In another preferred embodiment, carrageenan is used as the gellingagent. In a particularly preferred embodiment, carrageenan type II orany carrageenan which contains predominantly the iota form ofcarrageenan is used. In each embodiment, the cells to be tested aremixed in a suspension comprising a gelling agent, and then inoculatedinto a well, compartment, or other receptacle, which contains thebiochemical(s) to be tested, along with a gel-initiating agent such asvarious cations. Upon contact of the gelling agent with thegel-initiating agent (e.g., cations), the suspension solidifies to forma viscous colloid or gel, with the cells evenly distributed throughout.

The present invention contemplates a testing device that is a microplatesimilar in structure to microtiter plates ("microplates"or"MicroPlates™") commonly used in the art and commercially available fromnumerous scientific supply sources (e.g., Biolog, Fisher, etc.). Thus,in one embodiment, standard 96-well microtiter plates are used. In otherembodiments, microtiter plates with more wells are used (e.g., 384 welland 1536 well microtiter plates). Furthermore, the microtiter plateformat is suited for methods for kinetic analysis of substrateutilization by cells.

For example, in one embodiment, a test panel for detailed phenotypictesting of E. coli and S. typhimurium called the ES MicroPlate™ (Biolog)was used. This panel contains 95 carbon sources, which can be utilizedby most strains of these species. To perform a test, identical cellsuspensions of isogenic parental and mutant strains are prepared andpipetted into the 96 wells of a microplate. The cells are incubated forapproximately 16-24 hours and if a substrate oxidation occurs in a givenwell, a violet/purple color is produced due to coupled reduction of atetrazolium dye. Quantitation of the intensity of color is possiblethrough use of a microplate reader or comparable instrument, or theplates can even be compared by eye. For observation of differences at afiner level, the MicroPlates™ can be read at frequent time intervals todetermine the kinetics of color formation (i.e., carbon source oxidationrates) in each of the 96 wells. For a typical strain, perhaps 80 to 85wells provide positive reactions and useful data.

An alternate embodiment of the invention generally relates to a"microcard" (i.e., such as the MicroCard™ developed by Biolog) devicefor the multiparameter testing of chemical, biochemical, immunological,biomedical, or microbiological samples in liquid or liquid suspensionform in a small, closed, easy-to-fill device, and is particular suitablefor multiparameter testing and identification of microorganisms. It isnot intended that the present invention be limited to a particular sizeddevice. Rather, this definition is intended to encompass any devicesmaller than the commonly used, 96-well microplates. In one particularlypreferred embodiment, the microcard is approximately 75 mm in width and75 mm in length, and approximately 3 mm in depth. Approximatelyone-tenth the volume of cells are used to inoculate the compartments ofthe device, as compared to standard microplates. Indeed, the presentinvention contemplates a device comprising: a) a housing; b) a testingregion contained within the housing; c) a liquid receiving means on anexternal surface of the housing; d) a liquid flow-directing meansproviding liquid communication between the testing region and the liquidreceiving means; and e) a gas-venting, liquid barrier in fluidiccommunication with the testing region.

After the device has been filled, a non-venting, sealing tape can beapplied to the device to cover the gas-venting, liquid barrier to reducethe evaporation of the liquid from the device. In some embodiments, thetape can permit the molecular diffusion of oxygen and/or carbon dioxideinto or out of the device to maintain the desired chemical orbiochemical environment within the device for successful performance ofthe test. Where the liquid receiving means comprises liquid entry ports,a similar closing tape can be applied to close the port or ports toprevent spilling and evaporation of the liquid therefrom.

With any of the testing formats, the visual result that is detected byeye or by instrument can be any optically perceptible change such as achange in turbidity, a change in color, a change in fluorescence, or theemission of light, such as by chemiluminescence, bioluminescence, or byStokes shift. Color indicators may be, but are not limited to, redoxindicators (e.g., tetrazolium, resazurin, and/or redox purple), pHindicators, or various dyes and the like. Various dyes are described inU.S. Pat. Nos. 4,129,483, 4,235,964 and 5,134,063 to Barry R. Bochner,hereby incorporated by reference. See also B. R. Bochner, Nature 339:157 (1989); and B. R. Bochner, ASM News 55: 536 (1990). A generalizedindicator useful for practice of the present invention is also describedby Bochner and Savageau. See B. Bochner and M. Savageau, Appl. Environ.Microbiol., 33: 434 (1977).

Testing based on the redox technology is extremely easy and convenientto perform. A cell suspension is prepared and introduced into thetesting compartments of the device. Each compartment is prefilled with adifferent substrate.

In a preferred embodiment, all wells are prefilled with test formulacomprising a basal medium that provides nutrients for the cells, and acolor-change indicator, and each compartment is prefilled with adifferent carbon compound or "testing substrate," against which the cellis tested. "Basal medium," as used herein, refers to a medium whichprovides nutrients for the microorganisms or cells, but does not containsufficient concentrations of carbon compounds to trigger a colorresponse from the indicator. "Carbon compound," "carbon source" and"testing substrate" are equivalent terms, and are used interchangeablyherein to refer to a chemical (e.g., a carbon-containing compound) insufficient concentration as to trigger a color response from theindicator when it is utilized (metabolized) by a microorganism (e.g.,GN, GP, YT, and other MicroPlates™ commercially available from Biolog).In a particularly preferred embodiment, redox purple is used as a redoxindicator in the present invention.

One of the principal uses of the present invention is as a method anddevice for simple testing and speciation of microorganisms. The presentinvention contemplates microbiological testing based on the redoxtechnology discussed above wherein a sample of a pure culture ofmicroorganism is removed from a culture medium on which it has beengrown and suspended at a desired density in saline, water, gel, gellingagent, buffer, or solution (e.g., PPS) . This suspension is thenintroduced into the compartments of the testing device which have beenprefilled with basal medium, indicator, and substrate chemicals. Themethod is extremely easy and convenient to perform, and, unlike otherapproaches, the method and device do not require skilled personnel andcumbersome equipment.

In other preferred embodiments, the present invention involves the useof instruments such as the Biolog MicroStation™, an instrument systemthat allows the reading of testing panels inoculated with cells, andanalyzes the data obtained from the testing panels. This allows therapid analysis of multiple phenotypic characteristics for many celltypes (e.g., microbial strains) in a short time.

DEFINITIONS

The terms "sample" and "specimen" in the present specification andclaims are used in their broadest sense. On the one hand, they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, fecalmatter, cerebrospinal fluid (CSF), semen, and saliva, as well as solidtissue. These terms also refers to swabs and other sampling deviceswhich are commonly used to obtain samples for culture of microorganisms.

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water, and industrial samples, aswell as samples obtained from food and dairy processing instruments,apparatus, equipment, disposable, and non-disposable items. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

Whether biological or environmental, a sample suspected of containingmicroorganisms may (or may not) first be subjected to an enrichmentmeans to create a "pure culture" of microorganisms. By "enrichmentmeans" or "enrichment treatment," the present invention contemplates (i)conventional techniques for isolating a particular microorganism ofinterest away from other microorganisms by means of liquid, solid,semi-solid or any other culture medium and/or technique, and (ii) noveltechniques for isolating particular microorganisms away from othermicroorganisms. It is not intended that the present invention be limitedonly to one enrichment step or type of enrichment means. For example, itis within the scope of the present invention, following subjecting asample to a conventional enrichment means, to subject the resultantpreparation to further purification such that a pure culture of a strainof a species of interest is produced. This pure culture may then beanalyzed by the medium and method of the present invention.

As used herein, the term "culture" refers to any sample or specimenwhich is suspected of containing one or more microorganisms or cells. Inparticularly preferred embodiments, the term is used in reference tobacteria and fungi. "Pure cultures" are cultures in which the organismspresent are only of one strain of a particular genus and species. Thisis in contrast to "mixed cultures," which are cultures in which morethan one genus and/or species of microorganism are present.

As used herein, the term "organism" is used to refer to any species ortype of microorganism, including but not limited to bacteria, yeasts andother fungi. As used herein, the term fungi, is used in reference toeukaryotic organisms such as the molds and yeasts, including dimorphicfungi.

As used herein, the term "spore" refers to any form of reproductiveelements produced asexually (e.g., conidia) or sexually by suchorganisms as bacteria, fungi, algae, protozoa, etc. It is also used inreference to structures within microorganisms such as members of thegenus Bacillus, which provide advantages to the individual cells interms of survival under harsh environmental conditions. It is notintended that the term be limited to any particular type or location ofspores, such as "endospores" or "exospores." Rather, the term is used inthe very broadest sense.

As used herein, the terms "microbiological media" and "microbiologicalculture media," and "media" refer to any substrate for the growth andreproduction of microorganisms. "Media" may be used in reference tosolid plated media which support the growth of microorganisms. Alsoincluded within this definition are semi-solid and liquid microbialgrowth systems including those that incorporate living host organisms,as well as any type of media.

As used herein, the terms "culture media," and "cell culture media,"refers to media that are suitable to support the growth of cells invitro (i.e., cell cultures). It is not intended that the term be limitedto any particular cell culture medium. For example, it is intended thatthe definition encompass outgrowth as well as maintenance media. Indeed,it is intended that the term encompass any culture medium suitable forthe growth of the cell cultures of interest.

As used herein, the term "cell type," refers to any cell, regardless ofits source or characteristics.

As used herein, the term "cell line," refers to cells that are culturedin vitro, including primary cell lines, finite cell lines, continuouscell lines, and transformed cell lines.

As used herein, the terms "primary cell culture," and "primary culture,"refer to cell cultures that have been directly obtained from animal,plant or insect tissue. These cultures may be derived from adults aswell as fetal tissue.

As used herein, the term "finite cell lines," refer to cell culturesthat are capable of a limited number of population doublings prior tosenescence.

As used herein, the term "continuous cell lines," refer to cell culturesthat have undergone a "crisis" phase during which a population of cellsin a primary or finite cell line apparently ceases to grow, but yet apopulation of cells emerges with the general characteristics of areduced cell size, higher growth rate, higher cloning efficiency,increased tumorigenicity, and a variable chromosomal complement. Thesecells often result from spontaneous transformation in vitro. These cellshave an indefinite lifespan.

As used herein, the term "transformed cell lines," refers to cellcultures that have been transformed into continuous cell lines with thecharacteristics as described above. Transformed cell lines can bederived directly from tumor tissue and also by in vitro transformationof cells with whole virus (e.g., SV40 or EBV), or DNA fragments derivedfrom a transforming virus using vector systems.

As used herein, the term "hybridomas," refers to cells produced byfusing two cell types together. Commonly used hybridomas include thosecreated by the fusion of antibody-secreting B cells from an immunizedanimal, with a malignant myeloma cell line capable of indefinite growthin vitro. These cells are commonly cloned and used to prepare monoclonalantibodies.

As used herein, the term "mixed cell culture," refers to a mixture oftwo types of cells. In some embodiments, the cells are cell lines thatare not genetically engineered, while in other preferred embodiments thecells are genetically engineered cell lines.

As used herein, the terms "monolayer," "monolayer culture," and"monolayer cell culture," refer to cells that have adhered to asubstrate and grow in as a layer that is one cell in thickness.Monolayers may be grown in any format, including but not limited toflasks, tubes, coverslips (e.g., shell vials), roller bottles, etc.Cells may also be grown attached to microcarriers, including but notlimited to beads.

As used herein, the term "suspension," and "suspension culture," refersto cells that survive and proliferate without being attached to asubstrate. Suspension cultures are typically produced usinghematopoietic cells, transformed cell lines, and cells from malignanttumors.

As used herein, the term "carbon source" is used in reference to anycompound which may be utilized as a source of carbon for bacterialgrowth and/or metabolism. Carbon sources may be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, and peptides.

As used herein, the term "nitrogen source" is used in reference to anycompound which may be utilized as a source of nitrogen for bacterialgrowth and/or metabolism. As with carbon sources, nitrogen sources maybe in various forms, such as free nitrogen, as well as compounds whichcontain nitrogen, including but not limited to amino acids, peptones,vitamins, and nitrogenous salts.

As used herein, the term "sulfur source" is used in reference to anycompound which may be utilized as a source of sulfur for bacterialgrowth and/or metabolism. As with carbon and nitrogen sources, sulfursources may be in various forms, such as free sulfur, as well ascompounds which contain sulfur.

As used herein, the term "phosphorus source" is used in reference to anycompound which may be utilized as a source of phosphorus for bacterialgrowth and/or metabolism. As with carbon, nitrogen, and sulfur sources,phosphorus sources may be in various forms, such as free phosphorus, aswell as compounds which contain phosphorus.

As used herein, the term "antimicrobial" is used in reference to anycompound which inhibits the growth of, or kills microorganisms. It isintended that the term be used in its broadest sense, and includes, butis not limited to compounds such as antibiotics which are producednaturally or synthetically. It is also intended that the term includescompounds and elements that are useful for inhibiting the growth of, orkilling microorganisms.

As used herein, the term "testing substrate" is used in reference to anynutrient source (e.g., carbon, nitrogen, sulfur, phosphorus sources)that may be utilized to differentiate bacteria based on biochemicalcharacteristics. For example, one bacterial species may utilize onetesting substrate that is not utilized by another species. Thisutilization may then be used to differentiate between these two species.It is contemplated that numerous testing substrates be utilized incombination. Testing substrates may be tested individually (e.g., onesubstrate per testing well or compartment, or testing area) or incombination (e.g., multiple testing substrates mixed together andprovided as a "cocktail").

Following exposure to a testing substrate such as a carbon or nitrogensource, or an antimicrobial, the response of an organism may bedetected. This detection may be visual (i.e., by eye) or accomplishedwith the assistance of machine(s) (e.g., the Biolog MicroStationReader™). For example, the response of organisms to carbon sources maybe detected as turbidity in the suspension due to the utilization of thetesting substrate by the organisms. Likewise, growth can be used as anindicator that an organism is not inhibited by certain antimicrobials.In one embodiment, color is used to indicate the presence or absence oforganism growth/metabolism.

As used herein, the terms "chromogenic compound" and "chromogenicsubstrate," refer to any compound useful in detection systems by theirlight absorption or emission characteristics. The term is intended toencompass any enzymatic cleavage products, soluble, as well asinsoluble, which are detectable either visually or with opticalmachinery. Included within the designation "chromogenic" are allenzymatic substrates which produce an end product which is detectable asa color change. This includes, but is not limited to any color, as usedin the traditional sense of "colors," such as indigo, blue, red, yellow,green, orange, brown, etc., as well as fluorochromic or fluorogeniccompounds, which produce colors detectable with fluorescence (e.g., theyellow-green of fluorescein, the red of rhodamine, etc.). It is intendedthat such other indicators as dyes (e.g., pH) and luminogenic compoundsbe encompassed within this definition.

As used herein, the commonly used meaning of the terms "pH indicator,""redox indicator," and "oxidation-reduction indicator," are intended.Thus, "pH indicator" encompasses all compounds commonly used fordetection of pH changes, including, but not limited to phenol red,neutral red, bromthymol blue, bromcresol purple, bromcresol green,bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple,xylenol blue, methyl red, methyl orange, and cresol red. The terms"redox indicator" and "oxidation-reduction indicator" encompass allcompounds commonly used for detection of oxidation/reduction potentials(i.e., "eH" ) including, but not limited to various types or forms oftetrazolium, resazurin, methylene blue, and quinone-imide redox dyesincluding the compounds known as "methyl purple" and derivatives ofmethyl purple. The quinone-imide redox dye known as methyl purple isreferred to herein as "redox purple." In a particularly preferredembodiment, "redox purple" comprises the compound with the chemicalstructure shown in FIG. 5, VI. It is contemplated that analogousderivatives of the reagent (e.g., alkali salts, alkyl O-esters), withmodified properties (e.g., solubility, cell permeability, toxicity,and/or modified color(s)/absorption wavelengths) will be produced usingslight modifications of the methods described in Example 13. It is alsocontemplated that various forms of redox purple (e.g., salts, etc.), maybe effectively used in combination as a redox indicator in the presentinvention.

As used herein, the terms "testing means" and "testing device" are usedin reference to testing systems in which at least one organism is testedfor at least one characteristic, such as utilization of a particularcarbon source, nitrogen source, or chromogenic substrate, and/orsusceptibility to an antimicrobial agent. This definition is intended toencompass any suitable means to contain a reaction mixture, suspension,or test. It is intended that the term encompass microtiter plates, petriplates, microcard devices, or any other supporting structure that issuitable for use. For example, a microtiter plate having at least onegel-initiating agent included in each of a plurality of wells orcompartments, comprises a testing means. Other examples of testing meansinclude microtiter plates without gel-initiating means included in thewell. It is also intended that other compounds such as carbon sources orantimicrobials will be included within the compartments. The definitionis also intended to encompass a "microcard" or miniaturized plates orcards which are similar in function, but much smaller than standardmicrotiter plates (for example, many testing devices can be convenientlyheld in a user's hand). It is not intended that the present invention belimited to a particular size or configuration of testing device ortesting means. For example, it is contemplated that various formats willbe used with the present invention, including, but not limited tomicrotiter plates, microcards, petri plates, petri plates with internaldividers used to separate different media placed within the plate, testtubes, as well as many other formats.

As used herein, the term "gelling agent" is used in a broad genericsense, and includes compounds that are obtained from natural sources, aswell as those that are prepared synthetically. As used herein, the termrefers to any substance which becomes at least partially solidified whencertain conditions are met. For example, one gelling agent encompassedwithin this definition is Gelrite™, a gellan which forms a gel uponexposure to divalent cations (e.g., Mg²⁺ or Ca²⁺). Gelrite™ is producedby deacetylating a natural polysaccharide produced by Pseudomonaselodea, and is described by Kang et al. (U.S. Pat. Nos. 4,326,052 and4,326,053, herein incorporated by reference).

Included within the definition are various gelling agents obtained fromnatural sources, including protein-based as well as carbohydrate-basedgelling agents. One example is bacteriological agar, a polysaccharidecomplex extracted from kelp. Also included within the definition aresuch compounds as gelatins (e.g., water-soluble mixtures of highmolecular weight proteins obtained from collagen), pectin (e.g.,polysaccharides obtained from plants), carrageenans and alginic acids(e.g., polysaccharides obtained from seaweed), and gums (e.g.,mucilaginous excretions from some plants and bacteria). It iscontemplated that various carrageenan preparations will be used in thepresent invention, with iota carrageenan comprising a preferredembodiment. It is also contemplated that gelling agents used in thepresent invention may be obtained commercially from a supply company,such as Difco, BBL, Oxoid, Marcor, Sigma, or any other source.

It is not intended that the term "gelling agent" be limited to compoundswhich result in the formation of a hard gel substance. A spectrum iscontemplated, ranging from merely a more thickened or viscous colloidalsuspension to one that is a firm gel. It is also not intended that thepresent invention be limited to the time it takes for the suspension togel.

Importantly, it is intended that the present invention provides agelling agent suitable for production of a matrix in which organisms maygrow (i.e., a "gel matrix"). The gel matrix of the present invention isa colloidal-type suspension of organisms produced when organisms aremixed with an aqueous solution containing a gelling agent, and thissuspension is exposed to a gel-initiating agent. It is intended thatthis colloidal-type gel suspension be a continuous matrix mediumthroughout which organisms may be evenly dispersed without settling outof the matrix due to the influence of gravity. The gel matrix mustsupport the growth of organisms within, under, and on top of the gelsuspension.

As used herein the term "gel-initiating agent" refers to any compound orelement which results in the formation of a gel matrix, followingexposure of a gelling agent to certain conditions or reagents. It isintended that "gel-initiating agent" encompass such reagents as cations(e.g., Ca²⁺, Mg²⁺, and K+). Until the gelling agent contacts at leastone gel-initiating agent, any suspension containing the gelling agentremains "ungelled" (i.e., there is no thickening, increased viscosity,nor hardening of the suspension). After contact, the suspension willbecome more viscous and may or may not form a rigid gel (i.e., contactwill produce "gelling").

As used herein, the term "inoculating suspension" or "inoculant" is usedin reference to a suspension which may be inoculated with organisms tobe tested. It is not intended that the term "inoculating suspension" belimited to a particular fluid or liquid substance. For example,inoculating suspensions may be comprised of water, saline, or an aqueoussolution which includes at least one gelling agent. It is alsocontemplated that an inoculating suspension may include a component towhich water, saline or any aqueous material is added. It is contemplatedin one embodiment, that the component comprises at least one componentuseful for the intended microorganism. It is not intended that thepresent invention be limited to a particular component.

As used herein, the term "kit" is used in reference to a combination ofreagents and other materials. It is contemplated that the kit mayinclude reagents such as carbon sources, nitrogen sources, chromogenicsubstrates, antimicrobials, diluents and other aqueous solutions, aswell as microplates (e.g., GN, GP, ES, YT, SF-N, SF-P, and otherMicroPlates™, obtained from Biolog), inoculants, microcards, and platedagar media. The present invention contemplates other reagents useful forthe growth, identification and/or determination of the antimicrobialsusceptibility of microorganisms. For example, the kit may includereagents for detecting the growth of microorganisms followinginoculation of kit components (e.g.,tetrazolium or resazurin included insome embodiments of the present invention). It is not intended that theterm "kit" be limited to a particular combination of reagents and/orother materials. Further, in contrast to methods and kits which involveinoculating organisms on or into a preformed matrix such as an agarsurface or broth, the present invention involves inoculation of atesting plate in which the organisms are suspended within a gel-formingmatrix.

As used herein, the term "primary isolation" refers to the process ofculturing organisms directly from a sample. Thus, primary isolationinvolves such processes as inoculating an agar plate from a cultureswab, urine sample, environmental sample, etc. Primary isolation may beaccomplished using solid or semi-solid agar media, or in liquid. As usedherein, the term "isolation" refers to any cultivation of organisms,whether it be primary isolation or any subsequent cultivation, including"passage" or "transfer" of stock cultures of organisms for maintenanceand/or use.

As used herein, the term "presumptive diagnosis" refers to a preliminarydiagnosis which gives some guidance to the treating physician as to theetiologic organism involved in the patient's disease. Presumptivediagnoses are often based on "presumptive identifications," which asused herein refer to the preliminary identification of a microorganismbased on observation such as colony characteristics, growth on primaryisolation media, gram stain results, etc.

As used herein, the term "definitive diagnosis" is used to refer to afinal diagnosis in which the etiologic agent of the patient's diseasehas been identified. The term "definitive identification" is used inreference to the final identification of an organism to the genus and/orspecies level.

Although embodiments have been described with some particularity, manymodifications and variations of the preferred embodiment are possiblewithout deviating from the invention.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); TSA (trypticase soy agar); YME or YEME (Yeastextract-malt extract agar); EMB (eosin methylene blue medium); MacConkey(MacConkey medium); Redigel (RCR Scientific, Goshen, IN); Gelrite™(Merck and Co., Rahway, N.J.); Remel, (Remel, Lenexa, Kans.); Oxoid(Oxoid, Basingstoke, England); BBL (Becton Dickinson MicrobiologySystems, Cockeysville, Md.); DIFCO (Difco Laboratories, Detroit, Mich.,now part of Becton-Dickinson); U.S. Biochemical (U.S. Biochemical Corp.,Cleveland, Ohio); Fisher (Fisher Scientific, Pittsburgh, Pa.); Sigma(Sigma Chemical Co., St. Louis, Mo.); Biolog (Biolog, Inc., Hayward,Calif.); ATCC (American Type Culture Collection, Rockville, Md.); CBS(Centraalbureau Voor Schimmelcultures, Delft, Netherlands); CCUG(Culture Collection of University of Gothenberg, Gothenberg, Sweden);GSU (Georgia State University, Atlanta, Ga.); NRRL (USDA NorthernRegional Research Laboratory, Peoria, Ill.); and NCYC (NationalCollection of Yeast Cultures, Norwich, England); NCCLS (NationalCommittee for Clinical Laboratory Standards); API (API AnalytabProducts, Plainview, N.Y.); Flow (Flow Laboratories, McLean, Va.);bioMerieux (bioMerieux, Hazelwood, Mo.); and Molecular Devices(Molecular Devices, Mountain View, Calif.).

The following Tables list the principal bacterial strains used in thefollowing Examples, with Table 2 listing the various actinomycetes, andTable 3 listing other species of microorganisms.

                  TABLE 2                                                         ______________________________________                                        Actinomycetes Tested                                                               Organism         Source and Number                                       ______________________________________                                        Actinomadura ferruginea                                                                         USDA                                                           NRRL B-16096                                                                 Actinoplanes rectilineatus USDA                                                NRRL B-16090                                                                 Micromonospora chalcea USDA                                                    NRRL B-2344                                                                  Norcardiopsis dassonvillei USDA                                                NRRL B-5397                                                                  Saccharopolyspora hirsuta USDA                                                 NRRL B-5792                                                                  Streptomyces albidoflavus USDA                                                 NRRL B-1271                                                                  Streptomyces coeruleoribidus USDA                                              NRRL B-2569                                                                  Streptomyces griseus USDA                                                      NRRL B-2682                                                                  Streptomyces hygroscopicus USDA                                                NRRL B-1477                                                                  Streptomyces lavendulae USDA                                                   NRRL B-1230                                                                  Streptoverticillium salmonis USDA                                              NRRL B-1484                                                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Other Organisms Tested                                                             Organism          Source and Number                                      ______________________________________                                        Escherichia coli   ATCC #25922                                                  Staphylococcus aureus ATCC #29213                                             Providencia stuartii ATCC #33672                                              Pseudomonas cepacia ATCC #25416                                               Neisseria lactamica CCUG #796                                                 Xanthomonas maltophilia ATCC #13637                                           Vibrio metschnikovii ATCC #7708                                               Cedecea neteri ATCC #18763                                                    Rhodococcus equi ATCC #6939                                                   Dipodascus ovetensis ATCC #10678                                              Cryptococcus laurentii CBS #139                                               Cryptococcus terreus A CBS #1895                                              Kluyveromyces marxianus GSU #C90006070                                        Saccharomyces cerevisiae A NCYC ##505                                         Williopsis saturnus var. saturnus GSU #WC-37                                  Penicillium notatum ATCC #9179                                                Penicillium chrysogenum ATCC #11710                                           Rhizomucor pusillus ATCC #32627                                               Aspergillus niger ATCC #16404                                                 Tricophyton mentagrophytes ATCC #9129                                       ______________________________________                                    

EXAMPLE 1 Primary Growth of Actinomycetes

In this example, several attempts were made to grow variousactinomycetes in R2A liquid media prepared from the recipe of Reasonerand Geldreich (Reasoner and Geldreich, Appl. Environ. Microbiol., 49:1-7 [1985]), prior to preparation of inoculum suspensions forinoculating commercially available MicroPlates™ from Biolog (Biolog'sGN, GP, and YT MicroPlates™). This method proved unsuccessful andcumbersome. Also, it was virtually impossible to obtain uniform(homogenous) cultures of satisfactory quality.

Next, these organisms were grown on the surface of various agar media.It was thought this might provide a very simple means to harvest sporesfrom the culture, as the colonies tend to anchor into the agar matrixitself. The media used in this example included Sporulation Agar(described by R. Atlas in Handbook of Microbiological Media, CRC Press,Boca Raton, Fla., p. 834 [1993]), and YEME Agar with glucose omitted(described by E. B. Shirling and D. Gottlieb, in "Methods forCharacterization of Streptomyces Species," Int'l J. System. Bacteriol.,16: 313-330 [1966])(hereinafter referred to as YEMEWG).

Sporulation Agar (also known as m-Sporulation Agar) comprises agar (15g/l), glucose (10 g/l), tryptose (2 g/l), yeast extract (1 g/l), beefextract (1 g/l), and FeSO₄. 7H₂ O (1 μg/l), pH 7.2±0.2 at 25° C. Theseingredients are added to 1 liter of distilled/deionized water, and mixedthoroughly with heat to boiling. After the mixture has dissolved, it isautoclaved at 15 psi (121° C.) for 15 minutes, and dispensed intoplates.

YEMEWG Agar comprises Bacto yeast extract (4 g/l; Difco), and Bacto-maltextract (10 g/l; Difco). These ingredients are added to 1 liter ofdistilled/deionized water and mixed thoroughly. The pH is adjusted to7.3, and agar (20 g/l) is added to the mixture. The mixture is thenautoclaved at 121° C. for 15-20 minutes, and dispensed into Petri platesafter it is sufficiently cooled. YEMEWG was used because preliminarystudies indicated that, while glucose-containing YEME agar was adequatefor growth of the Streptomyces species, genera such as Nocardiopsis andActinoplanes grew better when glucose was omitted from the mediumrecipe.

Because of the interest in obtaining spores, media that encouragesporulation were tried. For example, YEMEWG was found to be particularlyuseful, as this medium gave satisfactory growth and sporulation of moststrains tested within 2-4 days of incubation at 26° C. Various agarconcentrations were tested during these preliminary studies, and it wasfurther observed that when YEMEWG was used, improved sporulationoccurred in the presence of a higher agar concentration (e.g. 25 g/l,rather than the 15 g/l, traditionally used in microbiological agarmedia).

This approach of growing actinomycetes on a sporulation-inducing mediumwould have the additional benefit of standardizing the physiologicalstate of the organisms, and would permit preparation of inoculaprimarily from spheroidal spores. It was usually a relatively simplematter to produce uniform, homogeneous suspensions containing spores.Occasionally, however, large clumps of the organisms and their aerialmycelia are obtained which do not readily disperse in solution. Whenclumps are formed, the suspension is allowed to sit for a few minutes,permitting the large fragments to settle to the bottom of the tube. Useof a light inoculum (i.e., a 1:10 dilution of an initial suspensionwhere the initial suspension has a transmittance level of 70%) alsohelps avoid problems with clumping of large fragments. Therefore, clumpscan be avoided in the preparation of the final inoculum because only asmall, clump-free aliquot of the initial suspension is used. For thoseorganisms that sporulate poorly, fragments of rods and/or mycelialfilaments were obtained from the agar surface in the same manner.

This example highlights the advantages of the present invention for theprimary growth and subsequent characterization of actinomycetes, incontrast to references that indicate growth of actinomycetes is veryslow. For example, Bergey's Manual® (T. Cross, "Growth and Examinationof Actinomycetes--Some Guidelines," in J. Holt et al., "TheActinomycetes," Bergey's Manual® of Determinative Bacteriology, 9th ed.,Williams & Wilkins, Baltimore, pp. 605-609 [1994]) indicates that"mature aerial mycelium with spores may take 7-14 days to develop, andsome very slow-growing strains may require up to 1 month's incubation."This is in stark contrast to the present invention, in which heavygrowth and sporulation is achieved within 2-4 days of incubation.

EXAMPLE 2 Preparation of Inoculum

In this experiment, a method more optimal for preparation of ahomogeneous inoculum was determined. For example, it was found that aneasy and reproducible method was to grow the organisms as described inExample 1 on YEMEWG- prepared with 25 g/l agar, or other suitable agarmedium. A low density inoculum (i.e., 0.01 to 0.1 OD₅₉₀) was thenprepared by moistening a cotton swab and rubbing it across the top ofthe colonies to harvest mycelia and spores. It was determined thatsterilized water and 0.85% sterile saline worked reasonably well as asuspension medium for all strains. However, some strains exhibited apreference for one or the other. For example, Streptomycescoeruleoribidus, S. hygroscopicus, and S. albidoflavus produced anaverage of ten additional positive reactions when water was used as thesuspension medium, whereas thirteen additional positive reactions wereobserved for S. lavendulae when saline was used as the suspensionmedium. The majority of the Actinomycetes performed better when waterwas used. Therefore, water was used routinely to prepare thesuspensions.

EXAMPLE 3 Preparation of Multi-Test Plates

The inocula prepared as described in Example 2 were used to inoculatevarious Biolog MicroPlates™, including the commercially available GN,GP, and YT MicroPlates™. A few strains worked well upon inoculation intothe GN or GP MicroPlates™ (e.g., S. lavendulae). However, for moststrains (e.g., A. ferruginea, and N. dassonvillei) no positive reactionswere observed. In addition, positive reactions were observed in all ofthe test wells for some organisms (e.g., S. hirsuta), indicating thatthere was a problem with false positive results.

Much improved results were obtained when the wells located in the bottomfive rows of the YT MicroPlate™ were used. It was thought that thisobservation was due to the absence of tetrazolium in these wells, as thetetrazolium present in the other wells appeared to inhibit the growth ofthe organisms. This was confirmed by testing the ability of theorganisms to grow on YEMEWG agar media containing various concentrationsof tetrazolium (20, 40, 60 and 80 mg/l). Many strains (e.g., S.coeruleoribidus, S. hygroscopicus, S. lavendulae, M. chalcea, N.dassonvillei, and A. rectilineatus) were inhibited at all of thesetetrazolium concentrations. Other organisms, such as S. griseus, S.albidoflavus, and S. hirsuta, were somewhat inhibited at the highertetrazolium concentrations, but grew in tetrazolium concentrations of 20and 40 mg/l.

Based on these experiments, MicroPlates™ were then tested that containedno tetrazolium (e.g., "SF-N" [GN MicroPlate™ without tetrazolium] and"SF-P" [GP MicroPlate™ without tetrazolium] MicroPlates™). These plateswere inoculated with water or saline suspensions of variousactinomycetes, and incubated at 26° C. for 1-4 days. Increased turbidity(i.e., growth of the organisms) was readable visually, or with amicroplate reader (e.g., a Biolog MicroStation Reader™, commerciallyavailable from Biolog), in as little as 24 hours for some strains. Forthe slow growing strains, growth was readable and the resultsinterpretable within 3-4 days, representing a significant improvementover the 7-10 day incubation period required using routine methods.

EXAMPLE 4 Use Of Gelrite™

Although growth was observable in the multi-test system described inExample 3, the results were still not completely satisfactory, due tothe unique growth characteristics of the actinomycetes. Many of thesestrains adhered to the plastic walls of the microplate wells, therebymaking detection of increased turbidity less than optimal. When theinoculating suspension is a liquid, turbidity often was concentratedalong the outer circumference of the wells, rather than producing auniform dispersion of turbidity throughout the wells.

In order to facilitate uniform dispersion of the inoculating suspensioncontaining organisms throughout the well, a gelling agent was added tothe suspension to prevent individual cells from migrating to the wellwalls. For example, preparations of Gelrite™ (commercially availablefrom Sigma, under this name, as well as "Phytagel") were found to behighly satisfactory. Gelrite™ does not form a gel matrix until it isexposed to gel-initiating agents, in particular, positively charged ionssuch as divalent cations (e.g., Mg²⁺ and Ca²⁺). As soon as the Gelrite™comes into contact with the salts present in the bottom of themicroplate wells, the gelling reaction begins and results in theformation of a gel matrix within a few seconds.

Various concentrations of Gelrite™ were tested, including 0.1, 0.2, 0.3,0.4, 0.5 and 0.6%. All concentrations gelled in the microplate, with thehigher concentrations producing a harder gel.

In view of the fact that most of the actinomycetes are obligate aerobes,there was a concern that the oxygen concentration within the gel must besufficient to permit growth. Thus, various gel depths were tested byusing 50, 100, or 150 μl suspensions of organisms in the wells. Each ofthese depths resulted in good growth of organisms, although it wasobserved that 0.4% Gelrite™ and an inoculum of 100 μl produced optimalresults, even with organisms such as Streptomyces lavendulae, a speciesthat is strongly hydrophobic and clings to the walls of wells when it issuspended in water. The 0.4% concentration of Gelrite™ was found toproduce an appropriate degree of viscosity to readily permit preparationof microbial suspensions and still be easily pipetted.

The entire procedure for growth and testing of the actinomycetesrequired a total of 3-7 days, including primary inoculation on YEMEWGmedium and other suitable media to determination and analysis of thefinal results. Importantly, a minimum amount of personnel time wasrequired (ie., just the few minutes necessary to inoculate the primarygrowth medium and then prepare the suspension for biochemical testing).Thus, the present invention provides a much improved means for the rapidand reliable identification of actinomycetes.

EXAMPLE 5 Comparison of Water and Gelrite™

In this Example, the eleven actinomycetes listed in Table 2 were testedin both water and gel suspensions. For each organism, a water suspensionof organisms with an optical transmittance of 70%, was diluted 1:10 ineither water or 0.4% Gelrite™. Thus, two samples of each organism wereproduced, one sample being a water suspension and one sample being asuspension which included Gelrite™.

One hundred microliters of each sample were inoculated into SF-PMicroPlates™ (GP MicroPlates™ without tetrazolium; commerciallyavailable from Biolog). The MicroPlates™ were incubated at 27° C. for 48hours, and observed for growth. As shown in the table below, the numberof positive reactions increased dramatically for the organisms suspendedin Gelrite™, as compared to water.

                                      TABLE 4                                     __________________________________________________________________________    Growth of Selected Streptomyces Species                                                    Number of                                                          Positive/Borderline Reactions Number of Positive/Borderline                   in Water Suspensions Reactions in Gel Suspensions                             (+/b) (+b/)                                                                 __________________________________________________________________________    Streptomyces coeruleorubidus                                                               5/35         35/25                                                 Streptomyces griseus 30/15   43/12                                            Streptomyces lavendulae 8/18 24/12                                          __________________________________________________________________________

EXAMPLE 6 Use of Resazurin

In this Example, three concentrations of resazurin dye (25 mg/l, 50mg/l, and 75 mg/l) were used as a redox color indicator of organismgrowth and metabolism. All of the eleven actinomycete strains listed inTable 2 were tested using these three concentrations of resazurin, and0.4% Gelrite™.

The expected color reaction, a change from blue to pink and eventuallyto colorless, as the dye is progressively reduced, occurred with alltest organisms after 48 hours of incubation at 27° C. This observationprovides a supplemental indicator of organism metabolism in addition toturbidity. No single resazurin concentration provided uniformly optimalresults. For example, N. dassonvillei produced a good differentialpattern of color change at 25 mg/l and 50 mg/l, whereas S. lavendulaeproduced false positive results (i.e., all colorless wells) at the lowerconcentrations (25 mg/l and 50 mg/l), but a good differential pattern ofcolor change at 75 mg/l.

Although the resazurin concentration may need to be adjusted dependingupon the organism tested, the use of resazurin as a color indicator mayprovide additional valuable information to characterize organisms at thespecies or strain level.

In the course of these experiments, it was also observed that pigmentsproduced by some actinomycetes in the various carbon sources tended tocreate very distinct and unique patterns. The unexpected observation wasmade that pigment production was enhanced by using a gel-formingsubstance in the inoculant.

Thus, different color patterns were obtained with the differingresazurin dye concentrations in combination with the natural pigmentsproduced. For example, at 50 mg/l resazurin, M chalcea produced a rangeof color intensities from colorless to light pink to bright pink andpurple. S. hygroscopicus produced a range of colors from yellow andorange, to colorless, pink and blue. Other species exhibited otherdistinct color patterns in the wells. This additional informationrelated to pigmentation and resazurin dye reduction, may be valuable totaxonomists and others interested in characterizing specific strainsand/or species of actinomycetes.

EXAMPLE 7 Use of Alternative Gelling Agents

Other gelling agents were tested in this Example. In addition toGelrite™, alginic acid, carrageenan type I, carrageenan type II, andpectin were tested for their suitability in the present invention. Allof these compounds are commercially available from Sigma.

Of these compounds, pectin was found to be unsuitable when tested byadding 1% pectin to SF-P MicroPlates™. Pectin has a yellowish cast toit, and is therefore not a colorless or clear compound. Furthermore,gelling was dependent upon the presence of sugars in the microplatewells. Because many of the substrates tested in this multitest format donot contain sugars, gelling did not occur uniformly in all wells.

All of these gelling agents with the exception of pectin, were testedwith the eleven actinomycetes listed in Table 2. The same MicroPlates™(SF-P), incubation time and temperature, as described in Example 5above, were used. The only variables were the different gelling agentsand varying concentrations of these agents.

The optimal viscosity and performance for each gelling agent wasdetermined. Optimal viscosity and performance was achieved at 1% alginicacid; 0.2% was optimum for both types of carrageenan; and 0.4% wasoptimum for Gelrite™. All of these gelling agents were also diluted tohalf the above concentrations and found to be useful even at these lowerconcentrations.

Overall, the results for Gelrite™ and carrageenan types I and II weresimilar, and the difference in gel concentration did not affect theresults significantly. However, the results for alginic acid were not asclearcut when the MicroPlates™ were observed by eye, as compared to theuse of an automatic plate reader (e.g., Biolog MicroStation Reader™,Biolog). Indeed, when read by eye, the results with alginic acid weresomewhat inferior to those obtained with Gelrite™. Carrageenan type IIwas slightly better than type I and it was also comparable to or betterthan Gelrite™. Surprisingly, the carrageenan type II functions aseffectively as the Gelrite™, although the carrageenan does not form arigid gel. This indicates that it is not necessary that a rigid gel beformed in order for the beneficial effects of these colloidal gellingagents to be observed.

EXAMPLE 8 Testing of Other Bacterial Species

In addition to the actinomycetes, the present invention is also suitablefor the rapid characterization of numerous and diverse organisms, suchas those listed in Table 3. The gram-negative bacteria tested covered arange of genera and tribes, including Pseudomonas cepacia, Providenciastuartii, Neisseria lactamica, Xanthomonas maltophilia, Vibriometschnikovii, Cedecea neteri, and Escherichia coli. Variousgram-positive bacteria were also tested, including Rhodococcus equi andStaphylococcus aureus.

These organisms were tested basically as described in Example 5 above,with GN MicroPlates™ (Biolog) used to test the gram-negative organisms,and GP MicroPlates™ (Biolog) used to test the gram-positive organisms.In addition, ES MicroPlates™ (Biolog) were also tested with some of thegram-negative species. Inoculation in 0.4% Gelrite™ was compared toinoculation in 0.85% saline. The inoculation densities used were thosenormally recommended for these MicroPlate™ test kits (55% transmittancefor the gram-negative organisms, and 40% for the gram-positiveorganisms). Following inoculation of the MicroPlates™ with 150 μlsuspensions of organisms in either saline or Gelrite™ per well, theMicroPlates™ were incubated at 35° C. for 16-24 hours.

All of these organisms performed well in the gel, with most producingbetter results in gel than in saline. For example, in the ESMicroPlate™, E. coli produced 43 positive reactions within 24 hours whenthe gel was used, but only 36 positive reactions when saline was used. Acorrect identification of C. neteri was obtained after only 4 hours ofincubation in the Gelrite™, whereas overnight incubation was requiredfor saline. Thus, a correct identification of this organism is possiblein a much shorter time period than the 24 hour incubation usuallyrequired for traditional testing methods.

In contrast to conventional biochemical testing materials and methodstraditionally used, the present invention often achieves a definitiveidentification in a significantly shorter time period.

EXAMPLE 9 Testing of Eukaryotic Microorganisms--Yeasts

This experiment was designed to determine the suitability of the presentinvention for use in identification of eukaryotic microorganisms, suchas yeasts. In this experiment, two types of reactions were observed toestablish a metabolic pattern: a) assimilation reaction tests which arebased on turbidity increases due to carbon utilization by the organisms;and b) oxidation tests, which also test for carbon utilization, butwhich detect utilization via a redox color change of the organismsuspension.

In this experiment, yeasts were first grown on BUY Agar (Biolog) a solidagar medium, and harvested from the agar surface as described in Example2 above. The organisms included in this example are listed in Table 3(D. ovetensis, C. laurentii, C. terreus, K marxianus, S. cerevisiae, andW. saturnus). Biolog YT MicroPlates™ (available commercially fromBiolog) were then inoculated with an inoculum having an opticaltransmittance of 50%, in either water or 0.4% Gelrite™. Each well of theYT MicroPlate™ was inoculated with 100 μl of either the water or 0.4%Gelrite™ suspension of organisms. Thus, there were two sets of 6MicroPlates™ each. The inoculated MicroPlatesm were incubated at 27° C.,and the results observed at 24, 48, and 72 hours of incubation.

With the oxidation tests, in most cases, the color changes developedmore rapidly in the plates with Gelrite™ used as the inoculant, comparedto the plates with water as the inoculant. For example, D. ovetensis, W.saturnus, K marxianus, and C. laurentii gave stronger reactions at 48hours with Gelrite™. In contrast, S. cerevisiae and C. terreus gavestronger reactions at 48 hours with water.

With the assimilation tests, in all cases the Gelrite™ was superior orequivalent to the water inoculant. The data shown in the Tables belowclearly demonstrate that more positive (+) and borderline (b) reactionswere obtained overall, when Gelrite™ was used.

                  TABLE 5                                                         ______________________________________                                        Positive (+) and Borderline (b) Reactions                                       After One Day of Incubation                                                                      Water   Gelrite ™                                       Organism (+/b) (+/b)                                                        ______________________________________                                        D. ovetensis      0/5    17/7                                                   K. marxianus 14/3 16/9                                                        W. saturnus  9/7 40/9                                                         C. terreus A  4/14 33/3                                                       C. laurentii 61/5 67/8                                                        S. cerevisiae A 24/5 22/2                                                   ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Positive (+) and Borderline (b) Reactions                                       After Two Days of Incubation                                                                     Water   Gelrite ™                                       Organism (+/b) (+/b)                                                        ______________________________________                                        D. ovetensis      9/2    22/2                                                   K. marxianus 14/5 39/4                                                        W. saturnus 23/7 46/5                                                         C. terreusA 21/7 45/4                                                         C. laurentii 65/0 77/3                                                        S. cerevisiae A 24/6 24/0                                                   ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Positive (+) and Borderline (b) Reactions                                       After Three Days of Incubation                                                                   Water   Gelrite ™                                       Organism (+/b) (+/b)                                                        ______________________________________                                        D. ovetensis     21/9    23/7                                                   K. marxianus 27/5 43/7                                                        W. saturnus 48/6 52/3                                                         C. terreus A 20/8 58/5                                                        C. laurentii 68/6 78/5                                                        S. cerevisiae A 24/8 24/2                                                   ______________________________________                                    

In these experiments, the surprising observation was made that someorganisms could be identified faster due to better growth (ie., growththat appeared much more rapidly and at a greater density), in the platewith the Gelrite™, as compared to the plate with water. For example,Dipodascus ovetensis developed a metabolic reaction pattern sufficientfor correct identification after 24 hours of incubation in the Gelrite™plate, while 48 hours of incubation was required to make the properidentification in the water plate.

In addition, many of the limitations and deficiencies of currentlycommercially available yeast identification systems, such as the Minitek(BBL), API 20C (API), expanded Uni-Yeast-Tek System (Flow), and Vitek(Biomerieux) were overcome or avoided in the present example (see e.g.,G. A. Land (ed.), "Mycology," in H. D. Isenberg (ed.), ClinicalMicrobiology Procedures Handbook, American Society for Microbiology, inparticular "Commercial Yeast Identification Systems," pp. 6.10.1 through6.10.5, [1994]). For example, in the Vitek system, heavily encapsulatedyeasts and isolates with extensive mycelial growth are sometimesdifficult to suspend. As indicated above, this limitation is avoided bythe present invention, allowing for reliable and reproducible testingprocedures and systems. In summary, the Gelrite™0 was shown to beclearly superior to water for the rapid identification of eukaryoticmicroorganisms.

EXAMPLE 11 Testing of Eukaryotic Microorganisms--Molds

This experiment was designed to determine the suitability of the presentinvention for use in identification of eukaryotic microorganisms, suchas molds.

In this experiment, the molds were first grown on modifiedSabouraud-Dextrose agar (commercially available from various sources,including Difco). This medium is prepared by thoroughly mixing dextrose(20 g/l), agar (20 g/l), and neopeptone (1 g/l) in 1 liter ofdistilled/deionized water. Heat is applied, until the mixture boils. Themedium is autoclaved for 15 minutes at 15 psi (121° C.). After cooling,the medium is distributed into petri plates.

The organisms included in this example are listed in Table 3 (P.notatum, P. chrysogenum, R. pusillus, A. niger and T. mentagrophytes).After they were grown on Sabouraud-Glucose agar, an inoculum wasprepared as described in Example 1. YT and SP-F MicroPlates™ (Biolog)were then inoculated with a 1:10 dilution of a starting inoculum havingan optical transmittance of 70%, in water, 0.2% carrageenan type II, or0.4% Gelrite™.

Each well of the SF-P MicroPlates™ was inoculated with 100 μl oforganisms suspended in either water, 0.2% carrageenan type II, or 0.4%Gelrite™. For the YT plates, 100 μl of organisms suspended in eitherwater, or 0.4% Gelrite™ were used to inoculate the wells. The inoculatedMicroPlates™ were incubated at 25° C., and the results observed by eyeand using a MicroStation Reader™ (Biolog) at 24 hour increments for atotal of 4 days of incubation.

In nearly all cases, the turbidity changes developed more rapidly in theplates with carrageenan or Gelrite™ used as the inoculant, compared tothe plates with water as the inoculant. The data shown in the Tablesbelow clearly demonstrate that for most organisms, more positive (+) andborderline (b) reactions were obtained overall, when carrageenan orGelrite™ was used, as compared to water. The results in these Tables arethose observed with the MicroStation Reader™ (Biolog).

It was also observed that the improvement in the results using Gelrite™or carrageenan as the gelling agent were sometimes more apparent whenthe test results were read visually, rather than by a machine (Biolog'sMicroStation Reader™). This was the case with T mentagrophytes, wherethe improved results obtained with carrageenan were in fact, alsoobtained with Gelrite™, although the reader did not detect thisaccurately at 72 hours. However, with longer incubation periods (e.g.,4-5 days), the visual and machine readings agree very well in nearly allcases.

                  TABLE 8                                                         ______________________________________                                        Positive (+)/Borderline (b) Reactions                                           After 72 Hours of Incubation in SF-P MicroPlates ™                                        Carrageenan Gelrite ™                                                                         Water                                       Organism (+/b) (+/b) (+/b)                                                  ______________________________________                                        P. notatum   54/11       52/14    47/11                                         P. chrysogenum 56/13 54/11 50/17                                              R. pusillus  4/13 5/5 2/6                                                     A. niger 23/17 29/12 17/10                                                    T. mentagrophytes 16/12 3/6 5/1                                             ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Positive (+)/Borderline (b) Reactions                                           After 72 Hours of Incubation in YT MicroPlates ™                                              Gelrite ™                                                                           Water                                             Organism (+/b) (+/b)                                                        ______________________________________                                        P. notatum       78/5     67/4                                                  P. chrysogenum 81/1 75/10                                                     R. pusillus  17/22 13/26                                                      A. niger 78/2 51/11                                                           T. mentagrophytes  2/1 2/1                                                  ______________________________________                                    

EXAMPLE 12 Antimicrobial Susceptibility Testing

In this Example, the suitability of a gel matrix for use inantimicrobial susceptibility testing was investigated. Two organisms,Staphylococcus aureus (ATCC #29213) and Escherichia coli (ATCC#25922)were tested against a panel of three antimicrobial agents: ampicillin,kanamycin, and tetracycline. All three antimicrobials were obtained fromSigma. Biolog's MT MicroPlates™ (Biolog), were used with 12.5 μl of a10% glucose solution added to each well. Kanamycin and tetracycline weredissolved in sterile water. Ampicillin was dissolved in phosphate buffer(pH 8.0)(0.1 M/l NaH₂ PO₄.H₂ O). For each antimicrobial agent, adilution series ranging from 0.25 μg/ml to 32 μg/ml final concentration,was prepared. A 15 μl aliquot of each dilution was pipetted into thewells of the MicroPlates™, with water used to dilute the kanamycin andtetracycline, and phosphate buffer (pH 6.0)(0.1 M/l NaH₂ PO₄.H₂ O) usedto dilute the ampicillin. For each MicroPlate™, a row of eight wellswithout antimicrobials was used as a control. In the MT MicroPlates™,tetrazolium is included as a color indicator. Unlike the actinomycetes,the most commonly isolated gram-negative and gram-positive bacteria arenot significantly inhibited by the presence of tetrazolium in theseMicroPlates™.

In addition to the MT MicroPlates™, Biolog's SF-N MicroPlates™ (GNMicroPlates™ without tetrazolium), and SF-P MicroPlates™ (GPMicroPlates™ without tetrazolium) were tested (all of these plates wereobtained from Biolog). E. coli was inoculated into the SF-NMicroPlates™, and S. aureus was inoculated into the SF-P MicroPlates™.In these MicroPlates™, 25 mg/l of resazurin was added as a colorindicator as an alternative to tetrazolium. In addition, 12.5 μl of 10%glucose solution and 15 μl of each antimicrobial dilution were added toeach well, as described in the paragraph above.

All of the wells in all of the MicroPlates™ were inoculated with 100 μlof a very light suspension (e.g., a 1:100 dilution of a 55%transmittance suspension of E. coli, and a 1:100 dilution of a 40%transmittance suspension of S. aureus), and incubated overnight at 35°C.

For each organism and each MicroPlate™, 0.85% saline and 0.4% Gelrite™were compared, by looking visually for the lowest antimicrobialconcentration that inhibited dye (tetrazolium or resazurin) reduction.The minimum inhibitory concentration (MIC) for each organism wasdetermined after 18 hours of incubation at 35° C. The MIC values foreach organism, as determined from these experiments, are provided in theTables below.

                  TABLE 10                                                        ______________________________________                                        MIC Determinations for E. coli                                                  in MT MicroPlates ™ Containing Tetrazolium                                 and SF-N MicroPlates ™ Containing Resazurin                                          Antimicrobial                                                     Diluent     Ampicillin Kanamycin Tetracycline                                 ______________________________________                                        Saline      1-2        16-32     0.5-1                                          Gelrite ™ 2-4  8-16 0.5-1                                                  NCCLS 2-8 1-4   1-4                                                           Expected Result                                                             ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        MIC Determinations for S. aureus                                                in SF-P MicroPlates ™ Containing Resazurin                                           Antimicrobial                                                     Diluent     Ampicillin Kanamycin Tetracycline                                 ______________________________________                                        Saline      1-4        16-32     0.25-2                                         Gelrite ™ 1-2 16-32 0.25-1                                                 NCCLS 0.25-1   1-4 0.25-1                                                     Expected Results                                                            ______________________________________                                    

As shown in these tables, the results in the Gelrite™ agreed with theresults obtained with saline as an inoculant within one two-folddilution. This is considered satisfactory according to the NationalCommittee on Clinical Laboratory Standards (NCCLS) guidelines (see e.g.,J. Hindler (ed.), "Antimicrobial Susceptibility Testing," in H. D.Isenberg (ed.), Clinical Microbiology Procedures Handbook, AmericanSociety for Microbiology, pp. 5.0.1 through 5.25.1, [1994]). In oneinstance, the MIC was slightly lower in saline as compared to Gelrite™.In three instances, the MIC's were slightly lower in Gelrite™, than insaline. Thus, the present invention provides a novel and usefulalternative method for determination of antimicrobial sensitivities ofmicroorganisms. Another advantage of this invention is that the test maybe conducted in a format that cannot be accidentally spilled.

EXAMPLE 13 Synthesis of Redox Purple

In this Example, the redox indicator referred to as "Redox Purple" wassynthesized for use in the present invention. In this Example, themethod of Graan et aL (T. Graan, et al., "Methyl Purple, anExceptionally Senstive Monitor of Chloroplast Photosystme I Turnover:Physical Properties and Synthesis," Anal Biochem., 144: 193-198 [1985])was used with modifications. This synthesis is shown schematically inFIG. 5 and the Roman numerals (i.e. I,II,III,IV and V) used in thisExample refer to those shown in FIG. 5. Unless otherwise indicated, thechemicals used in this Example were obtained from commercial sourcessuch as Sigma.

Briefly, the benzoquinone-4-chloroimide (FIG. 5, II) was produced bydissolving 5 g 4-aminophenol (FIG. 5, I) in 1 N aqueous HCl (75 mL) (0°C.), followed by the addition of 200 mL sodium hypochlorite (NaClO, 5%w/v) to produce a chloroimide derivative shown in FIG. 5, Panel A. Inthis reaction, the solution was continuously stirred and the temperaturemaintained below 4° C. during addition of the sodium hypochlorite. Afterstirring at room temperature for 12 hours, the yellow to orange coloredproduct was isolated by filtration, washed with cold distilled water anddried in air and in vacuo. In this step, the product was vacuum filteredusing a Buchner funnel, washed with a minimal amout of ice-cold water(approximately 30 ml) in the funnel, dried in air for approximately 24hours, and dried overnight in a vacuum dessicator.

The synthesis of 1-(3-hydroxyphenyl)-ethanol (FIG. 5, IV) was performedimmediately prior to its use, by the reduction of 5 g1-(3-hydroxyphenyl)-ethanone (available as m-hydroxyacetophenone fromTokyo Kasei kogyo Co., Ltd. Fukaya, Japan, with TCI America, inPortland, Oreg., being the U.S. distributor) (FIG. 5, III) in water (300mL) with sodium borohydride (NaBH₄, 1.5 g), as shown in FIG. 5, Panel B.The reaction was warmed as necessary to dissolve the starting materialand stirred until the evolution of H₂ ceased (approximately 1 hour). ThepH was decreased to 2.0 (i.e., with concentrated HCl) to remove excessborohydride, followed by addition of 150 ml saturated sodium borate.

The synthesis of redox purple was initiated by addition of thechloroimide derivative (II) to the freshly prepared solution of1-(3-hydroxyphenyl)-ethanol (IV), in borate buffer (Na2B₄ O₇ /H₃ BO₃).Sodium arsenite (NaASO₂, 10 g) (Sigma) was added to the reactionsolution, in order to promote the formation of the indophenol, as wellas minimize the occurrence of side reactions. This reaction solution wasstirred at room temperature for 2 hours, during which the blue color ofthe indophenol (FIG. 5, V) appeared. The reaction mixture was thenallowed to sit at room temperature for 7-8 days, during which theclosure of the heterocyclic ring was allowed to occur due to formationof an oxymethylene group bridge between the two phenolic residues of thequinone-imide. The ring closure was accompanied by a change in thesolution color to a dark purple.

The reaction mixture was filtered and the precipitate washed withminimal cold water as described above. The filtrate was saturated withan excess of solid sodium chloride (approximately 100 g), the solutionwas decanted off the excess salt on the bottom of the container, and thesolution extracted with diethylether (5×100 mL) until no moreorange-colored material was removed from the aqueous phase. Vigorousshaking of the ether and aqueous phases was avoided, as this was foundin some experiments to result in formation of an intractable emulsion.The combined ether layers were back-extracted with 70 mM aqueous sodiumcarbonate solution (25 mL), the pH of the sodium carbonate solutionreduced to 4.5 with glacial acetic acid, and the resulting mixturerefrigerated overnight at 4° C. The redox purple precipitated as thefree acid. Additional redox purple was obtained by acidifying theoriginal aqueous phases with glacial acetic acid (pH 4.5) and repeatingthe above purification. The total yield obtained by this synthesismethod was approximately 25%.

The purity of the redox purple synthesized according to this method was95-98%, as determined by thin-layer chromatography, a method that iswell know in the art (A. Braithwaite and F. J. Smith, in"Chromatographic Methods" Chapman and Hall [eds.], London [1985], pp.24-50.). It was found that the redox purple compound was not verysoluble in water as the free acid, but was quite soluble in slightlybasic solutions (e.g., 1 N NaHCO₃), or in organic solvents (e.g.methanol, ethanol, dimethyl sulfoxide [DMSO], dimethyl formamide [DMF],etc.). The compound was observed to be a deep purple color (i.e., ofapproximately 590 nm as an absorption wavelength) in basic solution andan orange-red color (470 nm) in acidic solution. It is contemplated thatanalogous derivatives of the reagent (e.g., alkali salts, alkylO-esters), with modified properties (e.g., solubility, cellpermeability, toxicity, and/or modified color(s)/absorption wavelengths)will be produced using slight modifications of the methods describedhere. It is also contemplated that various forms of redox purple (e.g.,salts, etc.), may be effectively used in combination as a redoxindicator in the present invention.

EXAMPLE 14 Redox Purple and E. coli Identification

In this Example, redox purple was used as the redox indicator in thetest system. E. coli 287 (ATCC #11775) was cultured overnight at 35° C.,on TSA medium supplemented with 5% sheep blood. A sterile, moistened,cotton swab was used to harvest colonies from the agar plate and preparesix identical suspensions of organisms in glass tubes containing 18 mlof 0.85% NaCl, or 0.2% carrageenan type II. The cell density wasdetermined to be 53-59% transmittance. One saline and one carrageenansuspension were used to inoculate Biolog GN Microplates™, with 150 μlaliquots placed into each well. The wells of this plate containtetrazolium violet as the redox indicator. Two ml of a 2 mM solution ofredox purple (sodium salt)(prepared as described in Example 13), or twoml of a 2 mM solution of resazurin (sodium salt) were added to the othertubes, to produce a final dye concentration of 200 μM. These suspensionswere used to inoculate Biolog SF-N Microplates™. As with the GNMicroplates™, aliquots of 150 μl were added to each well in the plates.The SF-N Microplates™ are identical to the GN MicroPlates™, with theexception being the omission of tetrazolium violet from the wells of theSF-N plates. The inoculated plates were incubated at 35° C. forapproximately 16 hours. The plates were then observed and the colors ofthe well contents recorded.

For the 0.85% NaCl and 0.2% carrageenan suspensions inoculated into theSF-N Microplate™, positive results were obtained for all three redoxindicators (ie., redox purple, tetrazolium violet, and resazurin) inwells containing the following carbon sources: dextrin, tween-40,tween-80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose,D-fructose, L-fucose, D-galactose, α-D-glucose, α-D-lactose, maltose,D-mannitol, D-mannose, D-melibiose, β-methyl-D-glucoside, L-rhamnose,D-sorbitol, D-trehalose, methyl pyruvate, mono-methyl succinate, aceticacid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid,D-glucuronic acid, α-ketobutyric acid, D,L-lactic acid, propionic acid,succinic acid, bromosuccinic acid, alaninamide, D-alanine, L-alanine,L-alanyl-glycine, L-aspargine, L-aspartic acid, glycyl-L-aspartic acid,glycyl-L-glutamic acid, D-serine, L-serine, inosine, uridine, thymidine,glycerol, D,L- α-glycerol phosphate, glucose-1-phosphate, andglucose-6-phosphate.

For the 0.85% NaCl and 0.2% carrageenan suspensions, negative resultswere obtained for all three redox indicators (i.e., redox purple,tetrazolium violet, and resazurin) in wells containing the followingcarbon sources: α-cyclodextrin, adonitol, D-arabitol, cellobiose,i-erythritol, xylitol, citric acid, D-glucosaminic acid,β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylaceticacid, itaconic acid, α-ketovaleric acid, malonic acid, quinic acid,sebacic acid, L-histidine, hydroxy L-proline, L-leucine, andD,L-carnitine. The negative control wells containing water, instead of acarbon source were also negative for all three redox indicators.

For glycogen, D-psicose, succinamic acid, and glucuronamide, negativeresults were obtained with both the 0.85% NaCl and carrageenansuspensions with redox purple. However, positive results were obtainedfor both suspensions with tetrazolium violet and resazurin.

For gentiobiose, m-inositol, cis-aconitic acid, L-phenylalanine,L-pyroglutamic acid, phenylethylamine, putrescine, 2-amino ethanol, and2,3-butanediol negative results were obtained with both the 0.85% NaCland carrageenan suspensions with redox purple and tetrazolium violet.However, positive/negative results were obtained with the 0.2%carrageenan suspension in resazurin, while the resazurin result with the0.85% NaCl was negative.

For lactulose, D-raffinose, formic acid, α-hydroxybutyric acid,L-glutamic acid, and L-proline, negative results were observed with the0.85% NaCl suspension tested with redox purple, although the remainingresults were positive.

For sucrose and L-ornithine, negative results were obtained for both the0.85% NaCl and 0.2% carrageenan suspensions tested with redox purple andtetrazolium violet. However, a negative result was observed for the0.85% NaCl suspension tested with resazurin and a positive result wasobserved for the 0.2% carrageenan suspension.

For turanose, both the 0.85% NaCl and 0.2% carrageenan suspensions werenegative when tested with redox purple, while the results for bothtested with tetrazolium violet were equivocal (+/-), the result for the0.85% NaCl suspension tested with resazurin was also equivocal (+/-),and the result for the 0.2% carrageenan tested with resazurin waspositive.

For α-ketoglutaric acid, negative results were observed for both the0.85% NaCl and 0.2% carrageenan suspensions tested with redox purple andtetrazolium violet, while positive results were observed for bothsuspensions tested with resazurin.

For D-saccharic acid, negative results were observed for both the 0.85%and 0.2% carrageenan suspensions tested with redox purple, while theresult with tetrazolium violet was equivocal (+/-) for 0.85% NaCl andnegative for carrageenan, and the result with resazurin was negative forthe 0.85% NaCl and positive for 0.2% carrageenan suspensions.

For L-threonine, equivocal (+/-) results were observed for 0.2%carrageenan suspensions tested with redox purple and tetrazolium violet,while the result with resazurin was positive. For the 0.85% NaClsuspension, the result was negative for redox purple, and positive fortetrazolium violet and resazurin.

For γ-aminobutyric acid and urocanic acid, negative results wereobserved for both the 0.85% NaCl and 0.2% carrageenan suspensions testedwith redox purple and tetrazolium violet, while equivocal (+/-) resultswere observed with 0.85% NaCl, and positive results were observed withthe 0.2% carrageenan.

In the inoculated GN Microplate™ (containing tetrazolium violet), thewells corresponding to the carbon sources utilized by E. coli 287 becameeither a light or dark purple, while the wells corresponding to thecarbon sources not utilized by this organism remained colorless. Incontrast, in the inoculated SF-N Microplate™ (containing redox purple),the color pattern was virtually reversed. For negative wells with redoxpurple, a blue to purple (i.e.,blue-purple, purple-tinged blue, orviolet) color was observed. In the SF-N Microplate™ plate, the wellscorresponding to carbon sources utilized by this organism were lightblue or were colorless, while the wells containing carbon sources notutilized by this organism remained dark blue. The color patterns wereeasily read and analyzed. Thus, the redox purple was shown to work in amanner that appears to be equivalent to tetrazolium violet for detectingcarbon source utilization by bacteria. However, there were three colorsobserved with the plates which included resazurin (i.e., blue, pink andcolorless), making the redox purple a more useful redox indicator, asthere was less ambiguity in the reading of the results.

The observation that none of the wells with redox purple was orange wasvery surprising, as the literature describing this compound indicatedthat there was an intermediate stage in the reduction of the dye whichwas expected to be reduced through the color progression of blue toorange to colorless. This two-stage reduction is in contrast to thetypical reaction observed with resazurin, which gives blue, pink, andcolorless wells when analyzed in a like manner. The side-by-side datafor the resazurin in this experiment, as well as other tests, confirmsthat it does form three colors. The degree to which the results of thevarious plates were in agreement are shown in the following Table.

                  TABLE 12                                                        ______________________________________                                        Comparison of Redox Purple and Resazurin                                        with Tetrazolium Violet                                                                            Number                                                    Dyes of Wells With Same                                                      Solution Compared Result (96 Wells/Plate) % Agreement                       ______________________________________                                        Saline                                                                              Redox Purple/                                                                              85/96         88.5                                            Tetrazolium Violet                                                           Gel Redox Purple/ 92/96 95.8                                                   Tetrazolium Violet                                                           Saline Resazurin/ 95/96 99.0                                                   Tetrazolium Violet                                                           Gel Resazurin/ 91/96 94.8                                                      Tetrazolium Violet                                                         ______________________________________                                    

The oxidized form of redox purple spectrally matches the reduced form oftetrazolium violet (i.e., with a maximum absorbance at 590 nm). This mayprovide an advantage, as detection methods such as spectrophotometrysettings may be used interchangeably with tetrazolium violet and redoxpurple.

EXAMPLE 15 Redox Purple and Identification of Fungi

In this Example, Aspergillus niger, Penicillium chrysogenum, andTrichoderma harzianum were tested using the redox purple redoxindicator.

First, the above named organisms were tested using the GN Microplate.However, none of these organisms reduced the tetrazolium violet in thewells of the plate. Thus, redox purple was investigated for use as analternative dye.

T harzianum DAOM 190830 was cultured for seven days at 26° C. on maltextract agar (Difco). A sterile, moistened cotton swab was used toharvest conidia from the culture and prepare a suspension in 16 ml of0.25% Gelrite™. The cell density was determined to be 75% transmittance.A 2 ml aliquot of a 2 mM solution of redox purple was added to thesuspension, along with 2 ml of 1 M triethanolamine-SO₄, pH 7.3. Thefinal concentration of redox purple was 200 μM, and the finalconcentration of triethanolamine-SO₄ was 100 mM. The final suspensionwas mixed well and used to inoculate the wells of a Biolog SF-NMicroplate™. In this Example, 100 μl of the suspension was added to eachwell. The inoculated SF-N Microplate™ was incubated at 30° C. forapproximately 24 hours, and observed.

For each carbon source utilized by the organism, the content of thewells was colorless. For each carbon source not utilized by theorganism, the content of the wells was blue. In this Example, for thisculture, positive results were obtained in the wells containing dextrin,glycogen, tween-40, tween-80, N-acetyl-D-glucosamine, L-arabinose,D-arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose,gentiobiose, α-D-glucose, D-mannitol, D-mannose, D-melibiose,β-methyl-D-glucoside, D-sorbitol, D-trehalose, methyl pyruvate,mono-methyl succinate, citric acid, D-galacturonic acid,β-hydroxybutyric acid, α-ketoglutaric acid, quinic acid, sebacic acid,succinic acid, bromo succinic acid, succinamic acid, L-alanine,L-alanyl-glycine, L-asparagine, L-glutamic acid, gylcyl-L-glutamic acid,L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine,γ-amino butyric acid, inosine, and glycerol.

EXAMPLE 16 Phenotype Analysis of E. coli

In this Example, ten strains of E. coli were tested and compared inBiolog ES MicroPlates™ and in Biolog MicroCards™ containing the samechemistry as the ES MicroPlates™. The strains tested in this Example arelisted in the following Table. As indicated by the designation "H?" inthis Table, the H antigen of some of the 0157 strains is unknown.

                  TABLE 13                                                        ______________________________________                                        E. coli STRAINS                                                                     Biolog Culture Number                                                                        Strain Name                                              ______________________________________                                        14443            MG1655 (FB426)                                                 14444 MG1655 xylA                                                             14445 MG1655 himA                                                              6320 W3110                                                                    6321 MG1655                                                                   6322 EMG2 (K12, F.sup.+)                                                     11547 O157:H7                                                                 13671 O157:H? gur+                                                            13673 O157:H?                                                                 13675 O157:H?                                                               ______________________________________                                    

All of the strains were cultured overnight on sheep blood agar plates(TSA with 5% sheep blood), at 35° C. Suspensions of the organisms wereprepared for testing using either PPS (0.01% phytagel, 0.03% pluronicF-58, and 0.45% NaCl) for MicroPlate™ testing, or IF1 (0.2% phytagel,0.03% pluronic F-68, and 0.25% NaCl) for MicroCard™ testing. All of thestrains were tested in both MicroCards™ and MicroPlates™. ForMicroPlate™ testing, inocula were prepared in PPS at a density of 63% T(as measured in the Biolog turbidimeter), in 20×150 mm tubes. ForMicroCard™ testing, inocula were prepared in IF1 at a density of 35% T(as measured in the Biolog turbidimeter) in 12×75 tubes. The inoculawere dispensed into MicroPlates™ (150 μl/well) or MicroCards™, asappropriate, and incubated at 35° C., for 24 hours. While results wereobtained using both the MicroPlates™ and MicroCards™, the results weremore consistent with MicroPlates™. Some wells in the MicroCard™ trappedair bubbles and gave false negative results. The MicroPlate™ results areindicated in Table 14, below, as well as described further in the textfollowing the Table. In Table 14, "+" indicates that the organism testedwas capable of utilizing the carbon source listed, while "-" indicatesthat the organism tested was not capable of utilizing the carbon sourcelisted, and "w" indicates weak positive reactions.

                                      TABLE 14                                    __________________________________________________________________________    Results for Ten E. coli Strains                                               Well                                                                             Carbon E. coli Strain                                                      No.                                                                              Source 14443                                                                             14444                                                                             14445                                                                             6320                                                                             6321                                                                             6322                                                                             11547                                                                             13671                                                                             13673                                                                             13675                              __________________________________________________________________________    A1 Water (control)                                                                      -   -   -   -  -  -  -   -   -   -                                    A2 L-arabinose + + + + + + + + + +                                            A3 N-acetyl-D- + + + + + + + + + +                                             glucosamine                                                                  A4 D-saccharic + + + + + + - - - -                                             acid                                                                         A5 Succinic acid + + + + + + + + + +                                          A6 D-galactose + + + + + + + + + +                                            A7 L-aspartic acid + + + - + + + + + +                                        A8 L-proline w - w + + + + + + +                                              A9 D-alanine + + + + + + + + + +                                              A10 D-trehalose + + + + + + + + + +                                           A11 D-mannose + + + + + + + + + +                                             A12 Dulcitol - - - - + - + + - +                                              B1 D-serine + + + + + + w - w w                                               B2 D-sorbitol + + + + + + - - - -                                             B3 Glycerol - - - + + + + + + +                                               B4 L-fucose + + + + + + + + + +                                               B5 D-glucuronic + + + + + + + + + +                                            acid                                                                         B6 D-gluconic + + + + + + + + + +                                              acid                                                                         B7 D,L-α-glycerol - - - - + + + + + +                                    phosphate                                                                    B8 D-xylose + - + + + + + + + +                                               B9 L-lactic acid + + + + + + + + + +                                          B10 Formic acid + + + + + + + + - +                                           B11 D-mannitol + + + + + + + + + +                                            B12 L-glutamic + - - - - w - + + +                                             acid                                                                         C1 Glucose-6- + + + + + + + + + +                                              phosphate                                                                    C2 D-galactonic + + + - + + - - - -                                            acid-γ-lactone                                                         C3 D,L-malic acid + + + + + + + + + +                                         C4 D-ribose + + + + + + + + + +                                               C5 Tween-20 - - - - w w w w w w                                               C6 L-rhamnose + + + + + + + + + w                                             C7 D-fructose + + + + + + + + + +                                             C8 Acetic acid + + + + + + + + + +                                            C9 α-D-glucose + + + w + + + + + +                                      C10 Maltose + - - + + + + + + +                                               C11 D-melibiose + + + + + + + + + +                                           C12 Thymidine + + + + + + + + + +                                             D1 L-asparagine + + + - + + + + + +                                           D2 D-aspartic acid - - - - - - - - - -                                        D3 D-glucosaminic - - - - - - - - - -                                          acid                                                                         D4 1,2-propanediol - - - - - - - - - -                                        D5 Tween-40 - - - w w w w w w w                                               D6 α-ketoglutaric + + + + + + - + + +                                    acid                                                                         D7 α-ketobutyric + + - + + - w - - -                                     acid                                                                         D8 α-methyl + + + + + + + + + +                                          galactoside                                                                  D9 α-D-lactose + + + + + + + + + +                                      D10 Lactulose - - - - - + + + + +                                             D11 Sucrose - - - - - - - + + +                                               D12 Uridine + + + + + + + + + +                                               E1 L-glutamine + + + - - + + + + +                                            E2 M-tartaric acid - - - - - - w + - -                                        E3 Glucose-1- + + + + + + + + + +                                              phosphate                                                                    E4 Fructose-6- + + + + + + + + + +                                             phosphate                                                                    E5 Tween-80 - - - w + w w w w w                                               E6 α-hydroxy- - - - - w - w - - w                                        glutaric acid γ-                                                        lactone                                                                      E7 α-hydroxy + + - + + + w w w w                                         butyric acid                                                                 E8 β-methyl + + + + + + + + + +                                           glucoside                                                                    E9 Adonitol - - - - - - - - - -                                               E10 Maltotriose + - - + + + + + + +                                           E11 2'-deoxy + + + + + + + + + +                                               adenosine                                                                    E12 Adenosine + + + + + + + + + +                                             F1 Glycyl-L- + + + + + + + + + +                                               aspartic acid                                                                F2 Citric acid - - - - - - - - - -                                            F3 M-inositol - - - - - - - - - -                                             F4 D-threonine - - - - - - - - - -                                            F5 Fumaric acid + + + + + + + + + +                                           F6 Bromo succinic + + + + + + + + + +                                          acid                                                                         F7 Propionic acid + + - + + + + + + +                                         F8 Mucic acid + + + + + + + + + +                                             F9 Glycolic acid + + - + + + - - - -                                          F10 Glyoxylic acid w w w + + + + - - -                                        F11 Cellobiose - - - - - - - - - -                                            F12 Inosine + + + + + + + + + +                                               G1 Glycyl-L- + + + + + + + + + +                                               glutamic acid                                                                G2 Tricarballylic - - - - - - - - - -                                          acid                                                                         G3 L-serine + + + + + + + + + +                                               G4 L-threonine + - - - - + - w w w                                            G5 L-alanine + + + + + + + + + +                                              G6 L-alanyl- + + + + + + + + + +                                               glycine                                                                      G7 Acetoactetic - - - w - - - - - -                                            acid                                                                         G8 N-acetyl-β-D- - - w w - + + w w +                                      mannosamine                                                                  G9 Mono-methyl + + + + + + + + + +                                             succinate                                                                    G10 Methyl + + + + + + + + + +                                                 pyruvate                                                                     G11 D-malic acid + + + + + + + + + w                                          G12 L-malic acid + + + + + + + + + +                                          H1 Glycyl-L- + + + + + + + + + +                                               proline                                                                      H2 P-hydroxy - - - - - - - - - -                                               phenylacetic                                                                  acid                                                                         H3 M- - - - - - - - - - -                                                      hydroxyphenyl                                                                 acetic acid                                                                  H4 Tyramine - - - - - - - - - -                                               H5 D-psicose + + + + + + + + + +                                              H6 L-lyxose - - - - + + - - - -                                               H7 Glucuronamide + + + + + + + + + +                                          H8 Pyruvic acid + + + + + + + + + +                                           H9 L-galactonic + + + + + + + + + +                                            acid γ-lactone                                                         H10 D-galacturonic + + + + + + + + + +                                         acid                                                                         H11 Phenylethyl - - - - - - - - - -                                            amine                                                                        H12 2-amino - - - - - - - - - -                                                ethanol                                                                    __________________________________________________________________________

Strains 14443 and 14444

Strain 14444 has been reported to be a xy1A (i.e., xylose-negative)mutant of strain 14443. The results of this experiment indicated thatwhile strain 14443 is xylose-positive (i.e., capable of utilizingxylose), strain 14444 is xylose-negative (i.e., incapable of utilizingxylose) However, strain 1444 was found to be negative also for maltose,maltotriose, L-proline, and L-threonine. While the results observed withL-proline and L-threonine may not be significant as these traits havebeen observed to be inconsistent between strains, the results obtainedwith maltose and maltotriose are significant, as discussed below.

Strains 14443 and 14445

Strain 14445 has been reported to be an himA mutant of strain 14443.Prior to this experiment, it was unknown what phenotypic changes due tothe himA allele, would be observed in 14445, as compared with strain14443. Differences between 14443 and 14445 were observed in eight tests.Strain 14445 was negative for utilization of maltose, maltotriose,α-ketobutyric acid, α-hydroxybutyric acid, propionic acid, glycolicacid, L-glutamic acid, and L-threonine. Although the results observedfor L-glutamic acid and L-threonine may not be significant, as thesetraits have been observed to be inconsistent between strains, theresults observed with maltose and maltotriose indicate the presence of adefect in maltose metabolism, as also observed in strain 14444. This wasconfirmed by contacting the source of these strains, Dr. Jeremy Glasner(in Dr. Fred Blattner's laboratory, at the University of Wisconsin), whotested these strains and confirmed that these strains had accidentallyacquired a maltose metabolism defect when he prepared a batch ofcompetent cells. Without the results of the present experiment, theaccidentally introduced defect would have gone unrecognized. With regardto the defects in utilization of the other four carbon sources, itappears that the himA allele may make cells deficient in utilization ofα-hydroxy acids, a new and surprising observation, that has beenheretofore unrecognized.

Strains 14443 and 6321

These strains are supposed to be the same strain, and both were obtainedfrom Dr. Barbara Bachmann, at the E. coli Genetic Stock Center. Prior totesting in this experiment, strain 14443 was maintained by Dr.Blattner's laboratory, while strain 6321 was stored at Biolog. Asindicated in Table 14, these two strains were shown to have differences,some of which may be insignificant, but some of which may have resultedfrom improper storage and maintenance, which caused the culture tochange over time.

Strains 6322, 6321, and 6320

Strain 6322 is the originating strain of the genetically important E.coli K12 culture. Strains 6321 and 6320 were reported as being derivedfrom 6322 via genetic manipulations that eliminated the lambda phage andF+ episome. Strain 6321 was created using careful genetic manipulations,and as indicated in Table 14, its pattern of carbon utilization observedin this experiment was very similar to that of strain 6322. However,strain 6320 was created through harsh treatment (exposure to X-rays),and it differs from strain 6322 in many traits.

Strains 11547, 13671, 1367, and 13675

These strains are all of the O157 serological line, and are consideredto be human pathogens. These strains are similar to each other, but arerather different from the K-12 strains. It is well known that most O157strains are sorbitol negative, and this was observed for these fourstrains. However, it was also found that these strains have otherspecial traits. For example, all four of these strains were alsonegative for D-saccharic acid, and D-galactonic acid-g-lactone. Inaddition, three of the four strains were positive for sucrose. Thenegative result observed for D-galactonic acid-g-lactone is particularlyinteresting. The genes involved in metabolism of D-galactonicacid-g-lactone (dgo) map at 82 minutes on the E. coli genome. Recentgenome sequencing data have indicated that in at least one O157 strain,a large "pathogenicity island" has been inserted in the E. coli genomeat 82 minutes. It is possible that the insertion of this pathogenicityisland may have resulted in the inactivation of the dgo genes.

EXAMPLE 17 Phenotypic Analysis of Yeast

In this Example, yeast are analyzed for phenotypic differences using theBiolog YT MicroPlate™. S. cerevisiae strains are grown on suitable media(e.g., as described in Example 9), and inoculated into the wells of theYT MicroPlate™ as described in Example 9. The ability of the strains toutilize different carbon sources (e.g., D-galactose) is then observedand compared, in order to assess the phenotypic differences between thestrains. As indicated in Example 9, water or Gelrite™ may be used as theinoculation suspension medium, as well as 0.85% NaCl or PPS (e.g., asdescribed in Example 16), above with 100 μl inoculated per well, ratherthan the 150 μl used with bacteria.

EXAMPLE 18 Kinetic Analysis

In this Example, two E. coli strains constructed so as to be isogenicwith the exception of a single allele are compared for their ability toutilize 95 different carbon sources in the Biolog ES MicroPlate™. Thestrains are cultured under identical conditions by growing them at roomtemperature on blood agar plates (TSA with 5% sheep blood). Suspensionsare prepared in PPS, as described in Example 16, above. Then, 150 μl ofthe suspensions are used to inoculate all of the wells of two ESMicroPlates™ (ie., one MicroPlate™ for each strain). The metabolicresponse (i.e., purple color formation) is followed kinetically at roomtemperature in a microplate reader (e.g., the Biolog MicroStation™) fora 24-hour period, and recorded, using SOFTmax®PRO software (MolecularDevices). Kinetic measurements are made using one of two methods. In thefirst method, each of the two MicroPlates™ are placed inside a kineticmicroplate reader and read at 15 minute intervals over a 24-hour period.In the second method, each of the two MicroPlates™ are cycled in and outof a microplate reader using a ROBOmax® in-feed stacking device(Molecular Devices). The MicroPlates™ are read at 15 minute intervalsover a 24-hour period. The kinetic readings are then converted into24-hour kinetic response patterns. The two patterns obtained arecompared, in order to identify differences in the organisms' responsesto each of the 95 carbon sources tested.

From the above Examples, it is clear that the present inventionrepresents an unexpected and much improved system for the broad-based,rapid biochemical testing and/or phenotypic testing of microorganismsand/or other cell types, in many uses and formats (or configurations).In one embodiment, the present invention provides a major advance in thetesting of actinomycetales, fungi, and other spore-formingmicroorganisms. The results are highly surprising in view of theobligate aerobic nature of most of these organisms. Using the novelapproach of embedding the organisms in a gel matrix, the biochemicaltest reactions are dispersed uniformly throughout the testing well,providing an easy to read indicator of organism growth and metabolism.In addition, both automated and manual systems with fixed time point orkinetic reading may be used in conjunction with the present invention.For example, the results may be observed visually (i.e., by eye) by theperson conducting the test, without assistance from a machine.Alternatively, the results may be obtained with the use of equipment(e.g., a microplate reader) that measures transmittance, absorbance, orreflectance through, in, or from each well of a multitest device such asmicroplate or microcard. Kinetic readings may be obtained by takingreadings at frequent time intervals or reading the test resultscontinuously over time.

In other embodiments, the present invention provides methods andcompositions for easily performing comparative testing of numerousphenotypes, thereby providing means to determine the functions ofvarious genes.

In summary, the embodiments of the present multitest gel-matrixinvention provide numerous advances and advantages over the prior art,including: (1) much greater safety, as there is no spillage, noraerosolization of cells, mycelia, nor spores, while performing orinoculating test wells; (2) faster biochemical reactions are produced,giving final results hours or days earlier than commonly used methods;(3) more positive biochemical reactions are obtained, giving a truerpicture of the microorganisms' metabolic characteristics; (4) darker,more clear-cut biochemical reactions and color changes are obtained; (5)more uniform color and/or turbidity are obtained, as the cells, mycelia,and/or spores do not settle and clump together at the bottom of thewells, nor do they adhere to the sides of the wells; (6) the reactionsare much easier to observe visually or with optical instruments (e.g.,the Biolog MicroStation Reader™); and (7) the overall process forperforming multiple tests is extremely simple and efficient, requiringvery little labor on the part of the microbiologist. All of theseadvantages enhance the speed and accuracy of scoring test results instudies to characterize and/or identify microorganisms, or to performcomparative phenotypic analysis of any cell type, including microbialstrains.

What is claimed is:
 1. A method for determining the phenotypedifferences in at least two cell preparations, comprising the stepsof:a) providing a testing device comprising a plurality of testingwells, wherein said wells contain a testing substrate and one or moregel-initiating agents; b) preparing a first suspension comprising afirst cell preparation, in an aqueous solution comprising a gellingagent, and a second suspension comprising a second cell preparation, inan aqueous solution comprising a gelling agent, under conditions suchthat said first and second suspensions remain ungelled; c) introducingsaid first and second suspension into said wells of said testing deviceunder conditions such that said first and second suspensions form a gelmatrix within said wells, such that said first and second cellpreparations are within said gel matrix; d) detecting the response ofsaid first and second cell preparations to said testing substrate; ande) comparing the response of said first and second cell preparations. 2.The method of claim 1, wherein said first and second cell preparationscomprise microorganisms selected from the group consisting of bacteriaand fungi.
 3. The method of claim 1, wherein said first and second cellpreparations contain cells of the same genus and species.
 4. The methodof claim 1, wherein said first and second cell preparations containcells that differ in one or more genes.
 5. The method of claim 1,wherein said gelling agent is selected from the group consisting ofGelrite™, carrageenan, and alginic acid.
 6. The method of claim 1,wherein said testing substrates are selected from the group consistingof carbon sources, nitrogen sources, sulfur sources, phosphorus sources,amino peptidase substrates, carboxy peptidase substrates, oxidizingagents, reducing agents, mutagens, amino acid analogs, sugar analogs,nucleoside analogs, base analogs, dyes, detergents, toxic metals,inorganics, and antimicrobials.
 7. The method of claim 1, wherein saidgel-initiating agent comprises cationic salts.
 8. The method of claim 1,further comprising a colorimetric indicator.
 9. The method of claim 8,wherein said colorimetric indicator is selected from the groupconsisting of chromogenic substrates, oxidation-reduction indicators,and pH indicators.
 10. The method of claim 9, wherein saidoxidation-reduction indicator is tetrazolium.
 11. The method of claim 9,wherein said oxidation-reduction indicator is redox purple.
 12. Themethod of claim 1, wherein said testing device is at least onemicroplate.
 13. The method of claim 1, wherein said response is akinetic response.
 14. A kit for determining the phenotypes of at leasttwo organisms, comprising:i) a microplate testing device containing aplurality of wells, wherein said wells contain one or moregel-initiating agents and one or more testing substrates; ii) a firstaqueous suspension comprising a gelling agent; and iii) a second aqueoussuspension comprising a gelling agent.
 15. The kit of claim 14, whereinsaid testing substrates are selected from the group consisting of carbonsources, nitrogen sources, sulfur sources, phosphorus sources, aminopeptidase substrates, carboxy peptidase substrates, oxidizing agents,reducing agents, mutagens, amino acid analogs, sugar analogs, nucleosideanalogs, base analogs, dyes, detergents, toxic metals, inorganics, andantimicrobials.
 16. The kit of claim 14, wherein said gelling agent isselected from the group consisting of Gelrite™, carrageenan, and alginicacid.
 17. The kit of claim 14, wherein said gel initiating agentcomprises cationic salts.
 18. The kit of claim 14, wherein said testingdevice further comprises a colorimetric indicator selected from thegroup consisting of chromogenic substrates, oxidation-reductionindicators, and pH indicators.
 19. The kit of claim 18, wherein saidoxidation-reduction indicator is tetrazolium.
 20. The kit of claim 18,wherein said oxidation-reduction indicator is redox purple.